Systems and methods for opening of a tissue barrier

ABSTRACT

Systems and methods for opening a tissue to a target value using microbubbles are disclosed herein. In an embodiment of a method for opening a tissue to a target value using microbubbles, a region of the tissue is targeted for opening, an acoustic parameter corresponding to the target value is determined, and an ultrasound beam is applied to the target region at the acoustic parameter such that the tissue at the target region is opened to the target value with the microbubbles. The acoustic parameter can be selected to control an acoustic cavitation event and, in some embodiments, controlling an acoustic cavitation event can include controlling a location, number and/or magnitude of acoustic cavitation events.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/US2010/049681, filed on Sep. 21, 2010, which isincorporated by reference in its entirety herein and from which priorityis claimed. This application claims priority to U.S. ProvisionalApplication Nos. 61/244,311 entitled “Improved Opening of TissueBarrier,” filed on Sep. 21, 2009, 61/353,611 entitled “Systems andMethods for Opening a Tissue Utilizing Certain Sonication Pulse Values,”filed on Jun. 10, 2010, and 61/353,631 entitled “Brain Drug DeliveryUsing Focused Ultrasound and Microbubbles,” filed Jun. 10, 2010, each ofwhich is incorporated by reference in its entirety herein and from whichpriority is claimed. This application is also related to U.S. patentapplication Ser. No. 12/077,612, filed Mar. 19, 2008, and InternationalPatent Application No. PCT/US09/056565, filed Sep. 10, 2009, each ofwhich is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under R01 EB009041 andR21 EY018505 awarded by the National Institutes of Health and CAREER0644713 awarded by the National Science Foundation. The government hascertain rights in the invention.

TECHNICAL FIELD

The present application relates to systems and methods for opening atissue utilizing acoustic parameters in conjunction with microbubbles.

BACKGROUND

Recent advances in molecular engineering and neuroscience have led to anincreasing number of biomarkers and therapeutic agents for themonitoring and treatment of neurological disorders. Many of these agentshave proven in vitro specificity or neurological potency, but their invivo efficacy remains limited by their inability to reach their targetdue to the blood-brain barrier. This interface regulates the exchange ofmolecules across the cerebral capillaries through passive, transport,and metabolic barriers, resulting in the exclusion of nearly all agentslarger than 400 Da from the brain's extracellular space. Biomarkers andtherapeutic agents, such as inhibitors to enzymes (˜1 kDa) andantibodies (30 to 300 kDa), are thus rendered ineffective because theydo not reach their intended targets.

SUMMARY

Systems and methods for opening a tissue to a target value are disclosedherein. Systems and methods for drug delivery across tissues, e.g.,across the blood brain barrier (BBB), are also disclosed herein.

In an embodiment of a method for opening a tissue to a target valueusing microbubbles, a region of the tissue is targeted for opening, anacoustic parameter corresponding to the target value is determined andan ultrasound beam is applied to the target region at the acousticparameter such that the tissue at the target region is opened to thetarget value with the microbubbles. The method can further includepositioning microbubbles in proximity to the targeted region and, insome embodiments, positioning the microbubbles can include performing aninjection of the microbubbles such that the microbubbles are positionedproximate to the targeted region. The method can further includedetermining a number of injections and/or a duration of an injectioncorresponding to the target value. In some embodiments, the injectioncan be a systemic injection, a bolus injection and/or a slow diffusioninjection. The acoustic parameter can be selected to control an acousticcavitation event and, in some embodiments, controlling an acousticcavitation event can include controlling a location, number and/ormagnitude of acoustic cavitation events. The acoustic parameter can be apulse length, a pulse repetition frequency, a burst length, a burstrepetition frequency, an ultrasound frequency, a pressure range, and/ora duration corresponding to the target value. In some embodiments, thepressure range can correspond to the resonance frequency of themicrobubbles proximate to the targeted region.

The method can include determining a concentration range of microbubblescorresponding to the target value and applying an ultrasound beam tomove the microbubbles into vessels of the tissue. In some embodiments,the microbubbles can have a size range of 1 to 10 microns, and in otherembodiments can have a size range of 1 to 2 microns, 4 to 5 microns, or6 to 8 microns. The microbubbles can be acoustically activated and/ormolecule-carrying. The molecule-carrying microbubbles can carry or becoated with medicinal molecules and/or a contrast agent and/or abiomarker and/or a liposome. Medicinal molecules and/or contrast agentscan also be separately positioned in proximity to the targeted region.

The method can further include imaging the targeted region, to form animage of the opened tissue. In some embodiments imaging the targetedregion includes applying an ultrasound beam to the targeted region,while in other embodiments imaging the targeted region includesutilizing a magnetic resonance imaging device and/or a fluorescenceimaging device to image the targeted region.

An embodiment of a system for opening a tissue to a target value using asolution of microbubbles having a size range corresponding to the targetvalue includes a targeting assembly for targeting a region of thetissue, an introducer for delivering the solution to a locationproximate to the targeted region and a transducer, coupled to thetargeting assembly, for applying an ultrasound beam to the targetedregion at an acoustic parameter corresponding to the target valuethereby opening the tissue with the microbubbles to the target value.The acoustic parameter can be selected to control an acoustic cavitationevent.

The system can further include an imaging device for capturing imagedata of the opened tissue of the targeted region, and a processor,operatively coupled to the imaging device, for processing the image datato form an image therefrom. In some embodiments the imaging deviceincludes a transducer for applying an ultrasound beam to the targetedregion, while in other embodiments the imaging device includes amagnetic resonance imaging device and/or a fluorescence imaging deviceto image the targeted region.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated and constitute part ofthis disclosure, illustrate some embodiments of the disclosed subjectmatter.

FIG. 1 illustrates a method for opening a blood-brain barrier in a brainof a subject to a target value in accordance with an exemplaryembodiment of the disclosed subject matter.

FIG. 2 illustrates a method for imaging the opening of a blood-brainbarrier in a brain of a subject to a target value in accordance with anexemplary embodiment of the disclosed subject matter.

FIG. 3(a) illustrates a system for opening and/or imaging the opening ofa blood-brain barrier in a brain of a subject to a target value inaccordance with an exemplary embodiment of the disclosed subject matter.

FIG. 3(b) illustrates another system for opening and/or imaging theopening of a blood-brain barrier in a brain of a subject to a targetvalue in accordance with an exemplary embodiment of the disclosedsubject matter.

FIGS. 4(a)-(e) illustrate a targeting system for locating a targetregion of the brain of a subject in accordance with an exemplaryembodiment of the disclosed subject matter.

FIG. 5 illustrates a system for opening the blood-brain barrier used inconnection with an experiment on mice in accordance with an exemplaryembodiment of the disclosed subject matter.

FIG. 6(a) illustrates a fluorescence image at ten times magnification ofa mouse brain after application of tissue opening techniques inaccordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 6(b)-(c) illustrate fluorescence images at four timesmagnification of a mouse brain after application of tissue openingtechniques in accordance with an exemplary embodiment of the disclosedsubject matter.

FIG. 7 is a graph illustrating the effects of varying the microbubbleconcentration in accordance with an exemplary embodiment of thedisclosed subject matter.

FIG. 8(a) is a graph illustrating the effects of varying the pulserepetition frequency in accordance with an exemplary embodiment of thedisclosed subject matter.

FIG. 8(b) is a graph illustrating the probability of blood-brain barrieropening as a function of varying the pulse repetition frequency inaccordance with an exemplary embodiment of the disclosed subject matter.

FIG. 9(a) is a graph illustrating the effects of varying the pulselength in accordance with an exemplary embodiment of the disclosedsubject matter.

FIGS. 9(b)-(c) are graphs illustrating the probability of blood-brainbarrier opening as a function of varying the pulse length in accordancewith an exemplary embodiment of the disclosed subject matter.

FIGS. 10(a)-(h) illustrate histological images of a mouse brain afterfocused ultrasound sonication with a varying pulse length in accordancewith an exemplary embodiment of the disclosed subject matter.

FIG. 11 illustrates an exemplary pulse and burst sequence in accordancewith an exemplary embodiment of the disclosed subject matter.

FIGS. 12(a)-(c) are graphs illustrating the effects of varying the burstrepetition frequency for three different pulse repetition frequencies inaccordance with an exemplary embodiment of the disclosed subject matter.

FIG. 12(d) is a graph illustrating the effects of varying the burstlength for a certain pulse repetition frequency and burst length inaccordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 13(a)-(i) illustrate images of a mouse brain subjected tosonication in accordance with an exemplary embodiment of the disclosedsubject matter.

FIGS. 14(a)-(e) illustrate images of a mouse brain subjected tosonication in accordance with an exemplary embodiment of the disclosedsubject matter.

FIGS. 15(a)-(d) illustrate images of a mouse brain subjected tosonication in accordance with an exemplary embodiment of the disclosedsubject matter.

FIGS. 16(a)-(e) illustrate images of a mouse brain subjected tosonication in accordance with an exemplary embodiment of the disclosedsubject matter. FIG. 16(f) is a diagram showing further details of thedisclosed subject matter.

FIGS. 17(i)(a)-(ii)(b) illustrate images of a mouse skull subjected tosonication in accordance with an exemplary embodiment of the disclosedsubject matter.

FIG. 18 illustrates images of a mouse brain subjected to sonication inaccordance with an exemplary embodiment of the disclosed subject matter.

FIG. 19 illustrates images of microbubbles in accordance with anexemplary embodiment of the disclosed subject matter.

FIGS. 20(a)-(c) are diagrams illustrating further features of thedisclosed subject matter.

FIG. 21 is a diagram illustrating further features of the disclosedsubject matter.

FIGS. 22(a)-(d) illustrate images of a mouse brain subjected tosonication in accordance with an exemplary embodiment of the disclosedsubject matter.

Throughout the figures and specification the same reference numerals areused to indicate similar features and/or structures.

DETAILED DESCRIPTION

The systems and methods described herein are useful for opening a tissueutilizing microbubbles and focused ultrasound at certain acousticparameters. Although the description provides as an the example openingthe blood-brain barrier, the systems and methods herein are useful foropening other tissues, such as muscular tissue, liver tissue or tumoroustissue, among others.

The subjected matter disclosed herein are methods and systems fordetermining the acoustic parameters for opening a tissue with theassistance of microbubbles to allow for the passage of certain moleculesover selected areas. Accordingly, the techniques described herein makeuse of selected acoustic parameters chosen to produce a desired openingeffect in a tissue when subjected to focus ultrasound utilizingmicrobubbles of selected sizes and in selected concentrations. Thetechniques described herein for determining the acoustic parameters foropening a tissue can also be employed in conjunction with otherultrasound techniques, e.g., diagnostic techniques, where opening of atissue should be avoided. The techniques described herein can be used todetermine the acoustic parameters that can be avoided in order toprevent unwanted tissue opening when utilizing such other techniquesthat, for example, use microbubbles. The techniques described hereinprovide for reversible opening of a tissue, e.g., the BBB.

In focused ultrasound (FUS), acoustic waves propagate severalcentimeters through water or tissue and converge onto a focal regionwhile its surroundings remain relatively unaffected. Noninvasive andlocalized drug delivery systems have emerged from advances in FUS andmicrobubble technologies. For example, techniques such as blood-brainbarrier (BBB) disruption for the treatment of neurological diseases,delivery of nanoparticles to tumors, gene therapy for treating heartconditions, and enhancement of renal ultrafiltration have all shownpromise due to their ability to increase uptake of luminal moleculesinto the interstitial space.

The mechanistic event underlying the tissue opening in such examples isthe reaction of microbubbles to ultrasonic pulses, which can result inan array of behaviors known as acoustic cavitation. In stablecavitation, the microbubble expands and contracts with the acousticpressure rarefaction and compression over several cycles, and suchaction can result in the displacement of the vessel diameter throughdilation and contraction. In inertial cavitation, the bubble can expandto several factors greater than its equilibrium radius and subsequentlycollapse due to the inertia of the surrounding media, thus also inducingan alteration of the vascular physiology. The type and magnitude of eachcavitation activity, including reversibility of the opening, can bedictated by, among other things, the microbubble composition anddistribution, the ultrasonic pulse shape and sequence, and the in vivoenvironment in which the bubbles circulate. Control of moleculardelivery using FUS can therefore be facilitated by selectingmicrobubbles and the acoustic environment conditions that they interactwith.

Generally, changing ultrasonic parameters has been associated with atradeoff between efficacy (e.g., high dose, homogeneous distribution,and consistency) and safety (e.g., erythrocyte extravasations andneuronal damage). The acoustic pressure can have a large influence onthe type and magnitude of acoustic cavitation activity. Thus, increasingthe pressure increases the likelihood and extent of BBB opening, butalso is associated with neurovascular and neuronal damage. At acousticpressures near the threshold of BBB disruption, histological assessmentsreveal no detectable damage (e.g., erythrocyte extravasations and/orneuronal damage) but at such low pressures there is also a reduction inmolecular delivery. Lowering the transmitted center frequency of theultrasound can result in a decrease of the acoustic pressure threshold.

The pulse repetition frequency (PRF) can affect the ability ofmicrobubbles to reperfuse the vasculature since each pulse can destroymicrobubbles. Further, it has been thought that a long pulse length (PL)was a necessary characteristic of an ultrasonic pulse for inducing BBBdisruption, for example PLs of 10 or 20 ms (15200 and 30400 cycles at1.5 MHz) and such long PLs have been associated with inhomogeneity ofdrug delivery. However, in accordance with an embodiment herein, it isshown that BBB disruption is feasible at a low pressure (less than 1MPa) using a PL of 33 μs (50 cycles at 1.5 MHz). Utilizing such a low PLand pressure, an improved distribution of the delivered agent wasobserved, but an associated decrease in concentration of moleculesdelivered was also observed. As further shown in an embodiment herein,pulse sequences based on the use of a PL of 2.3 μs (3.5 cycles at 1.5MHz) can enhance the dose and distribution of delivery withoutcompromising safety.

An acoustic parameter design can be formed on the basis that BBBdisruption is dependent on the number, magnitude and/or location ofcavitation events occurring throughout the cerebral microvasculature.Further, acoustic cavitation activity within the microvasculature can bemodified by taking into account concepts of microbubble persistence,fragmentation, and microvascular replenishment. In one embodiment, aseries of pulses can be grouped into a burst, with a sufficient durationbetween bursts to allow for microbubble replenishment in themicrovasculature before arrival of the subsequent acoustic pulses.Grouping pluses into bursts can increase the persistence and mobility ofthe microbubbles, which can result in a single bubble generatingcavitation activity at multiple sites along the cerebralmicrovasculature. Thus, in some embodiments, short PLs used in a burstsequence can enhance the dose and distribution of molecular deliverywithout attendant damage to the microvasculature. In the same or otherembodiments, the number of injections and duration of each injection ofbubbles can be altered to enhance the microbubble persistence, forexample.

FIG. 1 illustrates a method 100 for opening a tissue to a target value,e.g., a measure of increased ability of the tissue to pass moleculesthrough. The target value can be expressed in terms of an increase inthe size of vessels in the tissue, as an area of the tissue that hasbeen opened, or in terms of a rate at which molecules pass through,e.g., a permeability, or as a combination of any of these measures. Themethod 100 involves targeting 110 a region of the tissue for opening,determining 120 at least one acoustic parameter corresponding to thetarget value, positioning 140 microbubbles in proximity to the targetedregion, and applying 170 an ultrasound beam at the acoustic parameter tothe targeted region such that the tissue is opened with the assistanceof the microbubbles to the target value. In some embodiments,positioning 140 the microbubbles can include performing an injection ofthe microbubbles such that the microbubbles are positioned proximate tothe targeted region. The method 100 can further include determining anumber of injections and/or a duration of an injection corresponding tothe target value. In some embodiments, the injection 140 can be asystemic injection, a bolus injection and/or a slow diffusion injection.

As illustrated in FIG. 1, in one exemplary embodiment, the method 100can further include of determining 130 a concentration range ofmicrobubbles corresponding to the target value and the positioning 140of the microbubbles can also include positioning the microbubbles of theconcentration range that corresponds to the target value. The method 100can also include positioning 150 a contrast agent and/or medicinalmolecule (e.g., a drug) in proximity to the target region.

In one exemplary embodiment, method 100 can include applying 160 anultrasound beam to move the microbubbles into vessels of the tissue.This application 160 of the ultrasound beam can be the same, or adifferent, than the application 170 that is used to open the tissue.Further, the application 160 of the ultrasound beam can be at the same,or at a different, acoustic parameter than that determined 120 for thepurposes of opening the tissue.

The acoustic parameter to be determined 120 as corresponding to thetarget value can be selected to control one or more acoustic cavitationevents. The acoustic parameter(s) can be selected such that thelocation, number and/or magnitude of acoustic cavitation events can becontrolled in the targeted tissue. In some embodiments, the acousticparameter can be at least one of the pulse length, the pulse repetitionfrequency, the burst length, or the burst repetition frequency, or acombination thereof. In other embodiments, the acoustic parameters thatare determined 120 can be the pressure range, the frequency, and theduration of the application 170 of ultrasound.

In some embodiments, the target value of the tissue can be selectedbased on the size of the molecule that is to pass through the tissue,e.g., the BBB, or based on the size, e.g., area, of the region that isto be exposed to the molecule, or a combination of the two. Thus, insome embodiments, the acoustic parameters can be determined 120 suchthat the tissue is subject to a certain number of acoustic cavitationsat selected locations and of selected magnitudes, which can result in aselected number of molecules of a given size passing through the tissueat selected locations. In one exemplary embodiment, the target value canbe such that molecules up to the megaDalton size range are able to passthrough the BBB, e.g., 2 MDa molecules.

In one embodiment, the target value can be measured in terms of thenormalized optical density (NOD) of a contrast agent such as a dextran,e.g., Texas-Red® fluorescent dye and a molecular weight of 3 kiloDaltons(kDa), using the equation NOD=F_(L-ROI)−F_(R-ROI), where F_(L-ROI) isthe sum of the pixel values in left region of interest (ROI) of thebrain and R_(L-ROI) is the sum of the pixel values in the right ROI ofthe brain, and where the ultrasound was applied 170 to the left ROI.

In some embodiments, the acoustic parameter can be determined 120 byfinding the lowest acoustic parameter value for which the tissue willopen to the target value, where the target value is considered to be theminimum amount of opening. In one example involving murine brains, theacoustic parameters to be determined 120 were the PRF and the PL. Inaccordance with an exemplary experiment detailed below, a PRF value of 1Hz was experimentally determined 120 to be the lowest PRF for which theBBB of a mouse subject was observed to open based on an observed NOD ofapproximately 2×10⁷. A PL value of 0.033 ms was experimentallydetermined 120 to be the lowest PL for which the BBB was observed toopen based on an observed NOD of approximately 0.5×10⁷. In otherembodiments, a PL of 3 cycles (less than 2.5 μs) was experimentallydetermined 120 to open the BBB in mice, in an experiment conducted inaccordance with an exemplary embodiment described below.

In some embodiments, the acoustic parameter can be determined 120 byfinding the lowest reliable acoustic parameter value which will reliablyopen the tissue to the target value. In an example involving murinebrains where the target value was considered the minimum NOD for whichBBB opening was observed, the acoustic parameters to be determined 120were the PRF and the PL. In accordance with an exemplary experimentdetailed below, a PRF value of 5 Hz was experimentally determined 120 tobe the lowest PRF for which the BBB of a mouse subject was observed toreliably open based on an observed NOD of approximately 2×10⁷. A PLvalue of 0.2 ms was experimentally determined 120 to be the lowest PLfor which the BBB was observed to reliably open based on an observed NODof approximately 0.5×10⁷.

In yet other embodiments, the acoustic parameter can be determined 120by finding the acoustic parameter value above which no furthersignificant increase in opening of the tissue is achieved. In an exampleinvolving murine brains where the target value was considered to be theNOD above which no further increase in BBB opening was observed, theacoustic parameters to be determined 120 were the PRF and the PL. Inaccordance with an exemplary experiment detailed below, a PRF value of 5Hz was experimentally determined 120 to be the PRF for which no furthersignificant increase in the opening of the BBB of a mouse subject wasobserved and such opening corresponded to an NOD of approximately2.5×10⁷. A PL value of 10 ms was experimentally determined 120 to be thelowest PL for which no further significant increase in the opening ofthe BBB of a mouse subject was observed and such opening corresponded toan observed NOD of approximately 3×10⁷.

In the same or other embodiment involving murine brains, the acousticparameters to be determined 120 can be the burst repetition frequency(BRF) and burst length (BL), where each burst can represent a cluster ofpulses. A BRF of 10 Hz was experimentally determined 120 to open the BBBof a mouse subject, where the PRF was set at 100 kHz, and a BRF of 5 Hzwas experimentally determined 120 to produce the maximal BBB opening atthe same frequency. A BL of 100 pulses was experimentally determined 120to open the BBB where the PRF was set at 100 kHz and the BRF was set at5 Hz.

In some embodiments determining 130 a concentration range ofmicrobubbles corresponding to the target value can include determiningthe minimum microbubble concentration range that will open the tissue tothe target value. In an example involving murine brains, a microbubbleconcentration of 0.01 μl/g was experimentally determined 130 to be theminimum concentration needed to open the BBB. In same or anotherembodiment, the appropriate concentration of microbubbles can bedetermined 130 based on the nature of the subject, e.g., a human or amouse, based on the size of the target region, e.g., the surface area ofthe BBB that one wishes to open, and based on the vessel size in thetarget region, e.g., 4-8 μm, or a combination of these factors. In theexample of opening a BBB area on the order of millimeters, aconcentration range of 10⁷ to 10⁹ bubbles/mL can be appropriate. In oneexemplary embodiment, the total concentration for both size ranges ofbubbles, e.g., 1-2 and 4-5 μm, was kept constant at approximately8.5×10⁸ number of bubbles per mL. In order to ensure accuracy ofconcentration, the bubbles were generated at an initial yield largerthan the desired concentration and then diluted in PBS one minute beforeintravenous injection into the mouse.

In one exemplary embodiment, the bubble concentration can be chosen tobe the same across different size distributions as opposed to the volumefraction, because it can be assumed that BBB opening occurs discretely,e.g., the sites of molecular leakage highly correlated with theinstantaneous locations of the bubbles at the time of sonication. Thisimplies that BBB opening sites are punctuated along the length of thecapillaries. In the case where the volume fraction was kept the same forboth sets of bubbles, it is deemed that the imaging protocol used wouldhave the required sensitivity to detect minute increases influorescence.

As detailed in commonly assigned International Patent Pub. WO2010/030819, which is incorporated by reference in its entirety herein,in some embodiments the appropriate size range of microbubbles can bedetermined by comparing the bubble size to the cerebral vasculature sizeand selecting a bubble size that is small enough to perfuse the vesselswhile at the same time large enough to induce sufficient mechanicalstress on the vessel walls, such that the vessels are opened to thetarget value.

FIG. 2 illustrates a method 200 in accordance with the disclosed subjectmatter for imaging the opening of a tissue. The method 200 includes thesame basic techniques for opening the tissue to a target value:targeting 110 a region of the tissue for opening, determining 120 atleast one acoustic parameter corresponding to the target value,positioning 140 microbubbles of a known size range in proximity to thetargeted region, and applying 170 an ultrasound beam at the acousticparameter to the targeted region such that the tissue is opened with theassistance of the microbubbles to the target value. The method 200further includes imaging 210 the opened tissue. In some embodiments,imaging 210 the opened tissue can be the same as the application 170 ofan ultrasound beam to open the tissue. In another embodiment, imaging210 can include utilizing an MRI device to image the opening of thetissue.

FIG. 3(a) illustrates a system 300 for opening a tissue to a targetvalue. System 300 has many of the same features as the system describedin U.S. Patent Pub. No. 2009/0005711 and International Patent Pub. No.WO 2010/030819, commonly assigned patent applications, each of which isincorporated by reference in its entirety herein. Ultrasound waves aregenerated by a focused ultrasound transducer (FUS) 302, which can be asingle-element circular-aperture FUS transducer. In one exemplaryembodiment the FUS transducer 302 can be a single-element, sphericalsegment FUS transducer with center frequency of 1.525 MHz, a focal depthof 90 mm, an outer radius of 30 mm, and an inner radius of 11.2 mm(Riverside Research Institute, New York, N.Y., USA). The FUS transducercan be provided with hole in its center for receipt of an imagingtransducer 304, which can be a single-element diagnostic transducerhaving a center frequency of 7.5 MHz with a focal length of 60 mm(Riverside Research Institute, New York, N.Y., USA). The FUS transducer302 and the diagnostic transducer 304 can be positioned so that the fociof the two transducers are properly aligned, e.g., overlap.

Further illustrated in FIG. 3(a), an exemplary system 300 can include acone 306 filled with degassed and distilled water and mounted on system300. The cone 306 can, for example, be manufactured from a clearplastic, such as polyurethane. The water is contained in the cone 306 bycapping it with a material considered substantially “transparent” to theultrasound beam, such as an ultrathin polyurethane membrane 308 (Trojan;Church & Dwight Co., Princeton, N.J., USA).

The transducer assembly, which can include the FUS transducer 302 andthe diagnostic transducer 304, can be mounted to a computer-controlled3-D positioning system 310 (Velmex Inc., Lachine, QC, Canada), includingmotors VXM-1 and VXM-2 used in the exemplary embodiment. It isunderstood that other positioning systems can be incorporated forpositioning the transducer assembly with respect to the targeted tissue.

In the same or another exemplary embodiment, the FUS transducer 302 canbe driven by a function generator 320, e.g., function generatorHP33150A, manufactured by Agilent Technologies, Palo Alto, Calif., USA,through an amplifier 322, such as a 50-dB power amplifier 3100 L (ENI,Inc., Rochester, N.Y., USA). The diagnostic transducer 304 can be drivenby a pulser-receiver system 342, for example a pulser-receiver 5052 PR(Panametrics, Waltham, Mass., USA), connected to a digitizer 326, e.g.,digitizer CS14200 (Gage Applied Technologies, Inc., Lachine, QC,Canada). It is understood that the above-described components can bemodified or replaced with other components, as is known in the art, forproducing the ultrasound beams described herein. Computer 328 typicallyincludes a processor, such as a CPU (not shown), and can be anyappropriate personal computer or distributed computer system including aserver and a client. For example, a computer useful for this system is aDell Precision 380 personal computer. It is understood that any personalcomputer, laptop, or other processor that can load software andcommunicate with the various components discussed herein can be used. Amemory unit (not shown), such as a disk drive, flash memory, volatilememory, etc., can be used to store software for positioning andoperating the transducer assembly, image data, a user interfacesoftware, and any other software which can be loaded onto the CPU.

In another exemplary embodiment illustrated in FIG. 3(b), system 300′can include a transducer assembly having an array of a plurality ofsingle-element FUS transducers 304 and 305 which can be targeted todifferent regions of the tissue of the subject. Each FUS transducer 304,305 in the array can be fired individually, thereby permitting openingof the BBB in several locations without repositioning the transducerassembly.

Prior to sonication and in order to verify undistorted propagationthrough the skull, a scan, such as a 3-D raster-scan (lateral step size:0.2 mm; axial step size: 1.0 mm), of the beam of the FUS transducer 302,can optionally be performed in a large water tank containing degassedwater with a needle hydrophone having a needle diameter on the order ofabout 0.2 mm (Precision Acoustics Ltd., Dorchester, Dorset, UK). In thismanner the pressure amplitudes and three-dimensional beam dimensions ofthe FUS transducer 302 can be measured. The pressure amplitudes can bemeasured by calculating the peak-rarefactional pressure values andaccounting for an pressure attenuation due to transcranial propagation,e.g., an 18% pressure attenuation. The dimensions of the beam providedby the FUS transmitter 302 can have a lateral and axial full-width athalf-maximum (FWHM) intensity of approximately 1.32 and 13.0 mm,respectively, and in some embodiments can be approximately equal to thedimensions of the beam after propagation through the skull.

System 300 also includes a liquid container 334 containing anappropriate liquid 336, e.g., degassed and distilled water, which issealed at the bottom with a membrane 338, which can be a polyurethanemembrane that is acoustically and transparent, e.g., plastic wrap. Thesystem 300 can also include an optical imaging device 340, such as adigital camera, for imaging the skull of the subject 332 and a MRIdevice 350 for imaging the brain of the subject 332.

System 300 also includes a platform 330 for the subject. In oneexemplary embodiment, the platform 330 for the subject can be apolyurethane bed for a smaller subject 332, such as a mouse. In thisconfiguration, the membrane 338 can be placed over the subject 332. Inother embodiments, the platform 330 can be a hospital bed or surgicaltable, in which a larger subject 332 (such as a human subject) can belaid prone or supine and the transducer assembly positioned on top ofthe region of the skull targeted.

Additional components of the system 300 include a targeting system 400,coupled to the FUS transducer 302, for locating the focus of the FUStransducer 302 in the brain of the subject 332. The targeting system 400can be coupled by any known method that permits the targeting system 400to aid in properly targeting the FUS transducer 302 to the region ofinterest for opening of the target tissue, e.g., acoustic and/or opticalcoupling. FIGS. 4(a)-(d) illustrate a targeting system 400 for use withan embodiment where the subject 332 is a mouse. FIG. 4(a) illustratesmouse skull 401, where the skull's sutures can be seen through the skinand used as anatomic landmarks for targeting purposes. As illustrated inFIG. 4(a), the landmarks of mouse skull 401 include the sagittal suture402, the frontal bone 404, the interparietal bone 406, the left parietalbone 408, and the right parietal bone 410.

FIG. 4(b) illustrates the placement of targeting system 400 on skull 401in accordance with an exemplary embodiment. The targeting system 400 caninclude a plurality of members 420, 422, 424, such as thin metal bars,e.g., 0.3 mm thin metal bars, fabricated from an acoustically reflectivematerial, e.g., paper clips. The metal bars 420, 422, 424 can be placedon several landmarks of the skull of the subject to create a layout, orgrid. As illustrated in FIG. 4(b), a grid consisting of three equallyspaced 0.3-mm thin F2 metal bars 420, 422, 424 were placed in the waterbath 334 on top of the skull 401 and in alignment with these landmarks,e.g., bone sutures. The first bar 420 was aligned parallel and along thesagittal suture 402, and the second bar 424 was attached perpendicularlyto the first bar and in alignment with the suture between the parietal408 and interparietal bone 406. The third bar 422 was placed 4 mm awayfrom and parallel to the second bar 424.

FIG. 4(c) illustrates the location of a brain structure 440 to betargeted, here the hippocampus, relative to the landmarks noted above.The location of the hippocampi are assumed relative to the sutures basedon the mouse brain and known skull anatomy. In this exemplaryembodiment, using the grid positioning system 400, the location of oneof the hippocampi (indicated by circle 440) was reproducibly targetedwhen assumed to be at mid-distance (arrow 442) between the parallel bars422, 424 and 2 mm away from the center bar 420 (arrow 444).

To locate the desired brain structure 440 an image, such as a lateral2-D raster scan, of the grid configuration can be made using thediagnostic transducer 304. The focus of the FUS transducer 302 can thenbe positioned to precisely target the desired brain structure 440. Inanother exemplary embodiment, the targeting system can include otherimaging devices, such as a digital camera 340. For example, a digitalcamera 340 can be used to photograph the head of the subject 332. Therelevant landmarks can be identified in the photograph, and the focus ofthe FUS transducer 302 targeted to a location relative to the landmarks.In addition, other MRI targeting equipment, as is known in the art, canbe used for targeting the desired brain structure 440 or other targetedtissue structure.

FIG. 4(d) illustrates the actual location of the hippocampus 446 asindicated in the histology slice. FIG. 4(e) illustrates a lateral 2-Draster-scan 490 of the grid 400 using the diagnostic transducer 304. Thelocation of the hippocampus can be identified relative to this grid. Thefocus of the FUS transducer 302 was placed 3 mm beneath the top of theskull by measuring distance with the diagnostic transducer 304. Usingthe grid positioning system 400 and depth calculations, precise,accurate and reproducible targeting of the hippocampus or other brainstructures can be performed. In one exemplary embodiment, the gridpositioning system 400 allowed for sonication of the same location withgood accuracy across different mice. This allowed for not only goodreproducibility across different mice, but also a good comparison of BBBopening effects in different regions 440 within the sonicated area.

An exemplary method 100 for opening the BBB will be described inconnection with the above-referenced figures. The subject 332 ispositioned on a platform 330. Subject 332 can be positioned in a proneposition, and can be anesthetized for the sonication procedure. Thedegassed and distilled water bath 334 is suspended over the subject's332 head. Ultrasound gel can be used to reduce any remaining impedancemismatches between the thin plastic layer 338 and the subject's 332skin. The transducer assembly can be placed in the water bath 334 withits beam axis perpendicular to the surface of the skull 401.

The focus of the transducer is positioned inside the subject's 332brain. The focus can be targeted 110 to a region of the brain 440, suchas the desired brain tissue, e.g., the hippocampus 446, or to thevasculature of the brain, e.g., arteries, ventricles, arterioles, andcapillaries of the brain, or to other target tissue regions at differentlocations in the subject 332. The targeted region 440 of the brain canbe located 110 utilizing the targeting system as discussed above.

Example 1

FIG. 5 illustrates a system 300 used in an experiment, approved by theColumbia University Institutional Animal Care and Use Committee, onseventy-nine wild-type mice (strain: C57BL/6, mass: 28.0±4.5 g, sex:male; Harlan, Indianapolis, Ind., USA) which were studied in accordancewith the techniques described herein. As illustrated in FIG. 7, thesystem 300 can include a FUS transducer 302, a pulse-echo diagnostictransducer 304, a cone 306, a latex membrane 308, a 3-D positioningsystem 310 all operatively connected to a function generator 320, apower amplifier 322, a pulse-receiver system 324, a digitizer 326 and acomputer 327. The cone 306 can be inserted into a water container 334which is sealed at the bottom by a polyurethane membrane 338 and placedon the shaved skull 502 of the mouse subject 332. The mouse subject 332is held in place using a stereotaxic apparatus 504.

In the experiment, the mice were anesthetized using 1.25-2.50%isoflurane (SurgiVet, Smiths Medical PM, Inc., Wisconsin, USA)throughout both the BBB opening and transcardial perfusion procedures.After being anesthetized, each mouse 332 was placed prone with its headimmobilized by the stereotaxic apparatus 504 (David Kopf Instruments,Tujunga, Calif., USA). The hair on the skull was removed using anelectric trimmer and a depiatory cream. A degassed water-filledcontainer 334 sealed at the bottom with thin, acoustically and opticallytransparent, Saran™ Wrap 338 (Saran™; SC Johnson, Racine, Wis., USA) wasplaced on top of the mouse head 602 while ultrasound coupling gel wasused to eliminate any remaining impedance mismatch between the twosurfaces. The FUS transducer 302 was then submerged in the water of thecontainer 334 with its beam axis perpendicular to the surface of theskull 332.

The focus of the transducer was positioned inside the mouse brain usinga grid-positioning method that utilized the pulse-echo diagnostictransducer 304, as discussed above. The grid was constructed from three0.30 mm thin metal bars (i.e., paper clips) with two of the barsparallel to one another and separated by 4.00 mm. At the center of theparallel bars, and perpendicular to the two, was soldered the third bar.The grid was placed in the water bath 334, on top of the skull, and inalignment with sutures visible through the skin. The center bar wasaligned along the sagittal suture and one of the parallel bars with thelambdoid suture. A lateral two-dimensional raster-scan of the grid usingthe diagnostic transducer was made and the transducer's beam axis waspositioned 2.25 and 2.00 mm away from the sagittal and lambdoid suture,respectively. Finally, the focal point was placed 3.00 mm beneath thetop of the skull so that the acoustic wave propagated through the leftparietal bone and overlapped with the left hippocampus and a smallportion of the lateral region of the thalamus. The right hippocampus wasnot targeted and was used as the control. The grid positioning methodwas sufficiently precise to have the FUS beam consistently overlap thehippocampus of the murine brain.

The tissue opening procedure 100 involved injection 140, 150 a 25 μlbolus of Definity® microbubbles (1-10 μm) and a dextran contrast agent(Texas-Red® fluorescent dye with a molecular weight of 3 kDa) into thetail vein 1 minute after the start of sonication 170, with the injectiontaking place over a 30 second period. Sonication was performed for 11minutes total using pulsed FUS at a set pressure of 0.51 MPapeak-rarefactional at a single location (e.g., the hippocampus).

After the 11 minutes of sonication 170, the dextran was allowed tocirculate and accumulate in the mouse brain for 10 minutes, after whicha transcardial perfusion with phosphate buffer saline (138 mM sodiumchloride, 10 mM phosphate, pH 7.4) and 60 ml of 4% paraformaldehyde wasperformed. The brain was extracted from the skull and then post-fixed inthe paraformaldehyde overnight. Following the aforementioned procedures,the brain was prepared for frozen sections. The frozen sectioningprotocol provided an efficient means of analyzing fluorescence in orderto determine the threshold for BBB opening. In preparation of frozensectioning, the brain was cryoprotected by soaking it in 30% sucroseovernight. The brain was then embedded in a cutting temperature compound(Sakura Tissue-Tek O.C.T. Compound; Torrance, Calif., USA), frozen in asquare mold, and then sectioned using a cryostat into nine sections of100 μm slices in the horizontal orientation.

Bright field and fluorescent images of the frozen sections were acquiredusing an inverted light and fluorescence microscope (IX-81; Olympus,Melville, N.Y., USA) at 4× magnification and with a motorizedstage-scanner. Images of the paraffin sections were acquired using anupright light and fluorescence microscope (BX61; Olympus, Melville,N.Y., USA) at 4× and 10× magnification. The Texas Red-tagged dextranswere excited at 568±24 nm while emissions were filtered for 610±40 nm.

As noted above, the nine horizontal sections were chosen at definedcross-sections of the hippocampus. FIG. 6(a) illustrates a horizontalsection at 10× magnification, with the left and right ROIs shown in theleft and right boxes. FIG. 6(b) shows the left ROI, which was subjectedto sonication procedures, as detailed above, and FIG. 6(c) shows theright ROI, which was the control. In order to process the image, theregions of interest (ROIs) for each of the nine sections were outlinedusing Adobe® Photoshop® CS2 (San Jose, Calif., USA), as illustratedFIGS. 6(b)-(c). The outlines were then loaded into MATLAB® (Natick,Mass., USA) and used to isolate the hippocampus in the fluorescentimages. The images were normalized by dividing both the left and rightimages by the spatially averaged right (control) image of thehippocampus, thus calculating F_(L-HIP). The threshold for an image waswere the pixel value was greater than 2 standard deviations ofF_(L-HIP); images exceeding the threshold were excluded from thecalculations. The normalized optical density (NOD) was then calculatedfor each section, using the equation NOD=F_(L-ROI)−F_(R-ROI), whereF_(L-ROI) is the sum of the pixel values in left ROI of the brain andR_(L-ROI) is the sum of the pixel values in the right ROI of the brain.The NOD for the brain was calculated by averaging the NOD of across allnine sections. The resulting averaged NOD was then used to determinewhether, to what extent, the BBB had opened.

FIG. 7 is a graph illustrating the effects of varying the microbubbleconcentration in an exemplary embodiment where the FUS pressure was 0.46MPa, the microbubbles used where Definity® bubbles, the PRF was 10 Hzand the PL was 20 ms. The asterisks (*) indicates a significantdifference in NOD from the control. As can be seen in FIG. 7, there wasa significant NOD increase for all concentrations test and further therewas not a significant difference between the tested concentrations,indicating that with Definity microbubbles a concentration of 0.01 μl/gof body mass is both sufficient to open the BBB in mice and alsoreliable for doing the same.

FIG. 8(a) is a graph illustrating the effects of varying the PRF in anexemplary embodiment where the FUS pressure was 0.46 MPa, themicrobubbles used were Definity® bubbles, the microbubble concentrationwas set at 0.05 μl/g of body mass, and the PL was 20 ms. The asterisks(*) indicates a significant difference in NOD from the control. Asillustrated in FIG. 8(a), at least one pulse is needed to open the BBBin a mouse brain prepared in accordance with the procedures set forthabove. Further, the lowest PRF that was observed to open the BBB was 1Hz, while the lowest PRF that was observed to reliably open the BBB was5 Hz. FIG. 8(b) further illustrates these findings, showing the PRF as afunction of the probability of BBB opening. As illustrated in FIG. 8(b),at 5 Hz and above there is a 100% probability of the BBB opening in themice subjects prepared in accordance with the exemplary procedures setforth above. FIG. 8(a) also illustrates that no additional benefits aregained from using a PRF greater than 5 Hz.

FIG. 9(a) is a graph illustrating the effects of varying the PL in anexemplary embodiment where the FUS pressure was 0.46 MPa, themicrobubbles used were Definity® bubbles, the microbubble concentrationwas set at 0.05 μl/g of body mass, and the PRF was 10 Hz. The asterisks(*) indicates a significant difference in NOD from the control. Asillustrated in FIG. 9(a), lowest PL which resulted in BBB opening was0.033 ms, while 0.2 ms was the lowest PL which produced reliable BBBopening in mice prepared in accordance with the above-detailed exemplaryprocedures. The results illustrated in FIG. 9(a) demonstrate that,contrary to prior understanding, pulse lengths shorter than 10 ms canreliably open the BBB for FUS applications utilizing pressures less than0.5 MPa. FIG. 9(a) also illustrates that no additional benefits aregained from using a PL greater than 10 ms. FIGS. 9(b) and 9(c)illustrate the PL as a function of the probability of BBB opening, withFIG. 9(b) showing the data across all PLs tested in the exemplaryexperiment discussed herein and FIG. 9(c) illustrating the shortest PLstested. As illustrated in FIG. 9(c), a PL of as short as 0.1 ms has beenshown to reliably open the BBB of mice prepared in accordance with theexemplary procedures described herein, which is contrary to priorunderstanding of the necessary PL for FUS applications employingpressures less than 0.5 MPa.

FIGS. 10(a)-(h) are images of murine brains prepared in accordance withthe above-detailed exemplary procedures and illustrate the results ofvarying the PL from 0.033 ms (50 cycles) to 30 ms (45600 cycles). FIGS.10(a) and 10(b) illustrate the left sonicated ROI and right control ROI,respectively, at a PL of 0.033 ms (50 cycles). FIGS. 10(c) and 10(d)illustrate the left sonicated ROI and right control ROI, respectively,at a PL of 0.1 ms (152 cycles). FIGS. 10(e) and 10(f) illustrate theleft sonicated ROI and right control ROI, respectively, ata PL of 20 ms(30400 cycles). FIGS. 10(g) and 10(h) illustrate the left sonicated ROIand right control ROI, respectively, at a PL of 30 ms (45600 cycles).

FIG. 11 illustrates an exemplary pulse and burst sequence, where eachburst is composed set of pulses operating at a certain pulse ratefrequency (PRF). For example and as illustrated in FIG. 11, one pulsecan have a PL of 2.5 μs (approximately 3.5 cycles) at a pressure of 0.6MPa. As illustrated, the pulses can be repeated at a PRF of, forexample, 100 kHz. The pulsing can be continued for a period of time,e.g., 10 ms, which comprises a single burst of a certain length (BL).Finally, the bursts can be repeated at a certain rate, for example 50Hz, which is the BRF.

FIGS. 12(a)-(c) illustrate the NOD as a function of the BRF for threedifferent PRFs, for an exemplary experiment involving mice preformed inaccordance with the above-detailed procedures. FIG. 12(a) is a graphillustrating the effects of varying the BRF, in an exemplary embodimentwhere the FUS pressure was 0.46 MPa, the microbubbles used wereDefinity® bubbles, the microbubble concentration was set at 0.05 μl/g ofbody mass, the sonication duration was 11 minutes, the BL was 1000pulses, and the PRF was set at 100 kHz. FIG. 12(b) is a graphillustrating the effects of varying the BRF, in an exemplary embodimenthaving the same parameters as noted for FIG. 12(a), except at a PRF of25 kHz. FIG. 12(b) is a graph illustrating the effects of varying theBRF, in an exemplary embodiment having the same parameters as noted forFIG. 12(a), except at a PRF of 6.25 kHz.

FIG. 12(d) is a graph illustrating the effects of varying the BL, in anexemplary embodiment where the FUS pressure was 0.46 MPa, themicrobubbles used were Definity® bubbles, the microbubble concentrationwas set at 0.05 μl/g of body mass, the sonication duration was 11minutes, the BRF was 5 Hz, and the PRF was 100 kHz.

FIGS. 13(a)-(i) illustrate certain aspects of a mouse brain, prepared inaccordance with the exemplary methods described herein. Fluorescenceimages depicting delivery of dextrans at distinct molecular weights,spatially homogenous delivery, and outlines of neuronal axons are shown.The left ROI of the brain was sonicated in the presence of microbubblesand fluorescently-tagged (FIGS. 13(a) and 13(d)-(h)) 3-, (FIG. 13(b))10-, and (FIG. 13(c)) 70-kDa dextrans. Diffuse fluorescence regions canbe observed for all dextrans, whereas spots of high fluorescence areonly observed with the 70-kDa dextran. Pulsing in bursts using a3.5-cycle pulse length allowed for a homogeneous and diffuse spatialdistribution of 3-kDa dextran to the target ROI, shown in FIGS.13(d)-(f). FIG. 13(f) is a zoomed image of the white square in FIG.13(e), which is subsequently a zoomed image of the white square in FIG.13(d). As shown in the graph of FIG. 13(g), subregions within thehippocampus displayed difference in NOD. FIGS. 13(h) and 13(i) showthat, in some brains, the morphology of neurons and vessels can beobserved to have increased fluorescence over high levels of diffusefluorescence. FIG. 13(i) is a zoomed image of FIG. 13(h) using confocalmicroscopy. As shown, axons (white arrows) and a capillary (black arrow)are observed. The bars in FIGS. 13(a) and 13(i) depict 1 mm and 50 μm,respectively. An asterisk indicates an increase in left hemispheric NODper pixel (P<0.05) relative to the stratum pyramidale. The bar in FIG.13(a) depicts 1 mm, sr, stratum radiatum; sl: stratum lucidum; sp:stratum pyramidale; so: stratum orien.

Table 1 below illustrates the results of another experiment preformed onninety-nine male mice (strain: C57Bl6; 24.71±1.77 g) in accordance withthe exemplary methods described herein and the procedures of theColumbia University Institutional Animal Care and Use Committee. Table 1illustrates eighteen different experimental conditions, varying themicrobubble concentration (μl/g of body mass), the PRF (Hz), and the PL(ms). The results are shown in terms of the NOD (mean±s.d.×10⁹) and thenumber of mice with delivered dextran. The first entry corresponds to asham mouse, where no ultrasound was applied; in the second entry themicrobubbles and dextran were administered 1 minute before a 30-secondsonication; and in the final entry the microbubbles were injected over a180 second period. In the remaining entries the mice were intravenouslyinjected with a solution of dextran and microbubble 1 minute after thestart of an 11-minute sonication. All sonications were performed with an1.525 MHz acoustic beam and at a peak-rarefactional pressure of 0.46MPa.

TABLE 1 Microbubble Pulse concentration repetition Pulse NOD × 1e9Number of mice (μL/g of body frequency length (mean ± std. withdelivered mass) (Hz) (ms) dev.) dextran 0.05^(a) — — 0.01 ± 0.23 0/50.05^(b) 10 20 4.91 ± 0.94 5/5 0.05 10 20 4.45 ± 2.08 5/5 0.01 10 202.89 ± 1.99 5/5 0.25 10 20 5.34 ± 2.12 5/5 0.05 0.1 20 −0.17 ± 0.17  0/50.05 1 20 3.77 ± 4.52 3/5 0.05 5 20 4.21 ± 2.05 5/5 0.05 25 20 4.58 ±1.40 5/5 0.05 10 0.03 0.78 ± 1.41 2/5 0.05 10 0.1 1.20 ± 1.05 5/6 0.0510 0.2 0.98 ± 0.67 5/5 0.05 10 1 4.07 ± 3.71 5/5 0.05 10 2 1.84 ± 1.444/5 0.05 10 10 4.76 ± 2.03 5/5 0.05 10 30 5.77 ± 1.73 5/5 0.05 5 0.22.02 ± 1.38 5/5 0.05^(c) 5 0.2 1.58 ± 1.37 5/5 ^(a)Sham mouse. Noultrasound was applied. ^(b)Microbubble and dextran were administered 1minute before a 30-second sonication. ^(c)Microbubbles were injectedover 180 seconds.

Table 2 below illustrates the results of another experiment preformed onninety-five C57Bl6 male mice in accordance with the exemplary methodsdescribed herein and the procedures of the Columbia UniversityInstitutional Animal Care and Use Committee. Table 2 illustrateseighteen different experimental conditions, varying the PRF (kHz), BRF(Hz), the BL (# of pulses) and the peak-rarefactional pressure. Theresults are shown in terms of the NOD (mean±s.d.×10⁹) and the number ofmice with an incidence of NOD increase, calculated as detailed above.Those entries marked with an asterisk (*) correspond to sham mice, whereno ultrasound was applied and those entries marked with a doubleasterisk (**) correspond to parameters where pulses were emittedcontinuously and without bursts. For all entries the sonication was for11 minutes at a center frequency of 1.5 MHz, in the presence ofsystemically administered Definity® microbubbles (0.05 μl/g of bodymass) and fluorescently-tagged dextran (molecular weight: 3 kDa,fluorescent tag: Texas Red®) and the PL was 3.5 cycles (2.3 μs).

TABLE 2 Pulse Burst Burst repetition repetition length Peak- NOD × 1e9Incidence frequency frequency (# of rarefactional (mean ± std. of NOD(kHz) (Hz) pulses) pressure dev.) increase *— *— *— 0 0.02 ± 0.19 0/3100 **— **— 0.51 0.21 ± 0.32 1/3 100 10 1000 0.51 2.01 ± 1.44 3/3 100 51000 0.51 6.14 ± 1.45 3/3 100 2 1000 0.51 3.08 ± 2.76 3/3 100 1 10000.51 1.45 ± 0.73 3/3 100 0.1 1000 0.51 0.99 ± 0.85 2/3 25 **— **— 0.510.30 ± 0.20 1/3 25 10 1000 0.51 1.31 ± 1.35 2/3 25 5 1000 0.51 1.48 ±0.77 3/3 25 2 1000 0.51 3.34 ± 1.35 3/3 25 1 1000 0.51 1.92 ± 0.10 3/325 0.1 1000 0.51 0.35 ± 0.10 2/3 6.25 **— **— 0.51 −0.11 ± 0.35  1/36.25 5 1000 0.51 0.56 ± 0.95 2/3 6.25 2 1000 0.51 −0.16 ± 0.67  1/3 6.251 1000 0.51 0.78 ± 0.85 2/3 6.25 0.1 1000 0.51 0.68 ± 0.47 2/3 100 51000 0.37 0.60 ± 0.48 2/3 100 5 1000 0.25 0.10 ± 0.16 1/3 100 5 10000.13 0.10 ± 0.21 1/3 100 5 500 0.51 3.30 ± 0.58 3/3 100 5 100 0.51 3.60± 0.95 3/3 100 5 50 0.51 2.20 ± 0.54 3/3 100 5 10 0.51 2.14 ± 1.33 3/3100 5 5 0.51 0.13 ± 0.17 1/3 100 5 1 0.51 0.01 ± 0.11 0/3 *Sham micewhere no ultrasound was applied. **Parameters where pulses were emittedcontinuously and without bursts.

As illustrated in Table 2, emission of a continuous train of pulses at0.51 MPa and a PRF of 6.25, 25, and 100 kHz produced no significantincrease in NOD, which is a measure of the relative increase influorescence in the left (target) ROI relative to the right (control)ROI. Under each condition, only 1 out of 3 mice had an increase in NOD,and in these instances, the fluorescence was faint and distributed alongor near large vessels. Select intervals were evaluated by grouping 1000pulses into bursts and emitting them at a BRF of 0.1, 1, 2, 5, or 10 Hz,which corresponded to a burst repetition period (BRP) of 10, 1, 0.5,0.2, and 0.1 s, respectively. At a 100-kHz PRF, significant increases inNOD were observed at 1 and 5 Hz, while no increase was observed at 0.1,2, and 10 Hz. At a 25-kHz PRF, significant increases were observed at 1,2, and 5 Hz, while no increase was observed at 0.1 and 10 Hz. At a6.25-kHz PRF no significant increase was observed at any of the BRFsevaluated although some mice had observable increases in fluorescence.In general and as illustrated in Table 2, the NOD increased with theinterval between bursts and then decreased beyond a particular duration.Also, both the level and incidence of NOD decreased with the PRF. Themaximum average NOD increase was observed with a 100-kHz PRF and a 5-HzBRF.

Table 2 further illustrates the dependence of acousticpeak-rarefactional pressure on BBB disruption was evaluated in a sham (0MPa) and pressures of 0.13, 0.25, 0.37, and 0.51 MPa. A significantincrease in NOD was only observed at 0.51 MPa. Although 0.37 MPa had nosignificant increase in NOD, 2 out of 3 mice had detectable levels offluorescence. Therefore, the pressure threshold for BBB disruption for a3.5-cycle pulse was between 0.25 and 0.51 MPa. The effect of BL wasevaluated from 1 to 1000 pulses. A single pulse was insufficient indisrupting the BBB. The lowest pressure show feasible in disrupting theBBB was at 5 pulses and was observed in 1 out of 3 mice. A significantincrease in NOD was observed from 50 pulses and higher. In general,increasing the number of pulses increased the likelihood and magnitudeof NOD increase.

In an experiment using a 100-kHz PRF, a 5-Hz BRF, a 0.51 MPa pressureand a 1000 pulse BL, the relevance of the pulse-sequence topharmacologically-sized agents was evaluated with dextrans at molecularweights of 3-, 10-, and 70-kDa. Significant increases in NOD wereobserved using 3 and 70 kDa dextrans. The 10-kDa dextran wassuccessfully delivered in all 3 mice, but the increase was not assignificant (P=0.06). The 3-kDa agent was distributed most homogeneouslyand across a larger area than the other two molecular weights. Thedistribution of 10-kDa was diffuse as well, but did not extend spatiallyas far as the 3-kDa dextran. The 70-kDa dextran had heterogeneous spotsof high levels of fluorescence on top of diffusely distributedfluorescence. In certain instances, when high concentrations of 3-kDadextran were diffusely delivered to the target ROI, the morphology ofneurons and/or glial cells can be seen. For example and as illustratedin FIGS. 13(h) and 13(i), at a PRF of 100 kHz, a BL of 1000 pulses, anda BRF of 2 Hz, a neuronal axon with an approximately 1 μm diameter canbe observed extending from its cellular body and attached to acapillary, which had a diameter for approximately 4.5 μm.

Example 2

In another exemplary experiment, the efficacy of the FUS methoddescribed herein is shown by accomplishing two objectives after BBBopening: 1) diffusion of brain-derived neurotrophic factor (BDNF) acrossthe BBB and containment of BDNF within the targeted region, i.e., thehippocampus and 2) associated activation of the BDNF receptor and itsdownstream signaling molecules in neurons, illustrating bioactivity ofthe functional BDNF upon delivery.

To visualize the passage of BDNF across the BBB, the fluorescent dyeAlexa Fluor 594 was conjugated to BDNF prior to the experiments. Thefluorescent tag is expected to not modify the transport properties ofBDNF given its relatively small molecular weight (˜0.3 kDa) compared tothe BDNF's (27 kDa). Following successive application of FUS sonication(for example as shown in FIGS. 4(c) and 5), microbubble injection, andintravenous injection of BDNF, the mice (n=3) were sacrificed 20-30 minpost-sonication for histological analysis 1) to allow for the BDNF toaccumulate in the BBB-opened region and facilitate detection of thecompound (higher fluorescent intensity) at the sonicated areas; 2) toallow the compound to circulate through the microvasculature beforebeing cleared by the venous system, since circulating BDNF has ahalf-life of less than 10 min; and 3) to allow for the downstreamsignaling cascade to be activated. The activation of the BDNF receptor,TrkB, can occur within seconds of BDNF delivery.

Animals

A total of seven C57Bl6 male mice were used for this study (17.6-23.0 g,Harlan Laboratories). The animals were anesthetized with a mixture ofoxygen (0.8 L/min at 1.0 Bar, 21° C.) and 1.5-2.0% vaporized isoflurane(Aerrane, Baxter Healthcare) using an anesthesia vaporizer (SurgiVet,Smiths Group). The mouse's vital signs were monitored continuously andisoflurane was adjusted throughout the experiment as needed. TheColumbia University Institutional Animal Care and Use Committee (IACUC)gave approval for the mouse studies.

The total number of mice used in the BDNF study was seven (n=7) asfollows: three mice were injected with BDNF, sonicated and sacrificedafter 20-30 min for both IHC & fluorescence analysis, one mouse wasinjected with BDNF, sonicated and sacrificed but died after 3 min (IHCand fluorescence analysis were also performed but discounted in thestatistical analysis) and three controls were sonicated and sacrificedafter 20-30 min (No BDNF) for IHC analysis. Three types of “controls”were used to show the effect of sonication on the BDNF permeationthrough the blood-brain barrier and subsequent activation of thedownstream signaling cascade as follows:

a) to demonstrate the difference between the left (sonicated)hippocampus and the right (non-sonicated) hippocampus in four mice(three mice sacrificed after 20-30 min, one died after 3 min) followingBDNF injection and sonication;

b) to demonstrate no difference between the left (sonicated) hippocampusand the right (non-sonicated) hippocampus in three control micefollowing only sonication (no BDNF) so as to rule out the effect ofsonication alone on signaling cascade activation; and

c) to obtain negative controls for the IHC analysis in all studied mice,where no primary antibody was added to the sections.

The statistical analysis was based on the three BDNF-injected mice thatwere sacrificed after 20-30 min, and did not include the 3-min mousecase. The results for the case of 3-min BDNF-injected mouse are alsopresented for comparison in terms of the downstream signaling activationand its time dependence in order to support previous reports on thetemporal sequence of signaling cascades downstream of BDNF. The BDNF wasinjected into the femoral vein 10-min after sonication while the micewere still anesthetized. There was at least 3 min (1 mouse) and 20-30min (3 mice) of circulation before PBS transcardial perfusion wasstarted.

Neurotrophic Factors

Brain-Derived Neurotrophic Factor (BDNF)

The Brain-Derived Neurotrophic Factor (BDNF) conjugated to Alexa Fluor®594 dye was used (Invitrogen Corp, Carlsbad, Calif., USA). BDNF HumanRecombinant was produced in Escherichia Coli and is a homodimer,non-glycosylated, polypeptide chain containing 2×119 amino acids with atotal molecular mass of 27 kDa. According to the supplier, it waspurified by proprietary chromatographic techniques and the sequence ofthe first five N-terminal amino acids was determined and found to beMet-His-Ser-Asp-Pro. Biological activity was determined by evaluatingED50 (50 ng/ml), calculated by the dose-dependent induction of ACHE(acetylcholine esterase) in rat basal forebrain primary septal culture.The compound (6.0 mg) was custom conjugated to Alexa Fluor® 594 dye(˜1:1 molar ratio) and provided in a fine lyophilized powder. The vialswere stored under −18° C. until use.

Glial-Derived Neurotrophic Factor (GDNF) and Neurturin (NTN)

A total of 12 mice received FUS followed by GDNF (40-90 mg/kg in 0.15 mlPBS, n=10), as well as NTN (20 mg/kg in 0.2 ml PBS, n=2) injections. Twomice were used for the NTN study. Both GDNF and NTN were conjugated withAlexa Fluor® 488 fluorescent dye. Four sites within a 1 mm square in thecaudate were sonicated at a frequency of 1.5 MHz, with a pulse length of15,000 cycles (n=3) and 30,000 cycles (n=8), at varying pressures.Detailed acoustic parameters were shown in Table 3.

TABLE 3 FUS parameters used in the case of GDNF and NTN Compound GDNFNTN PL (cycles) 15,000 30,000 30,000 P-N pressure 0.30 0.45 0.60 0.300.45 0.60 0.60 Circulation time 45 180 180 60 60 6. 4 3 6 6 30

In the case of 6.5 minutes circulation time, blood was drawn after 45seconds to confirm the circulation and fluorescence of the protein.Brain, liver, kidney, and testes were extracted and fixed for frozensection. Organs were then frozen into blocks and sectioned at 100 μm topotentially locate GDNF.

Ultrasound

A single-element spherical segment FUS transducer (center frequency:1.525 MHz; focal depth: 90 mm) was driven by a function generator(Agilent Technologies) through a 50-dB power amplifier (ENI) to generatetherapeutic ultrasound waves. A pulse-echo transducer (center frequency:7.5 MHz; focal length 60 mm) was positioned through a center hole of theFUS transducer so that the foci of the two transducers were aligned. Itwas driven by a pulser-receiver system (Panametrics) connected to adigitizer (Gage Applied Technologies) and was used for imaging. A conefilled with degassed and distilled water and capped with an acousticallytransparent polyurethane membrane was mounted on the transducer system(FIG. 5). The transducers were attached to a computer-controlled 3Dpositioning system (Velmex). The FUS transducer's pressure amplitudereported in this Example was previously measured with a needlehydrophone (needle diameter: 0.2 mm; Precision Acoustics) in degassedwater while accounting for 18.1% attenuation by the mouse skull. Thedimensions of the beam were measured to have a lateral and axialfull-width at half-maximum (FWHM) intensity of approximately 1.32 and13.0 mm, respectively.

Targeting Procedure

The head of each anesthetized mouse was immobilized using a stereotaxicapparatus. The fur on top of the head was removed with an electric razorand a depilatory cream. After applying ultrasound gel, a water bath withits bottom made of an acoustically and optically transparent membranewas placed on top of the head and gel. A grid positioning method totarget the mouse hippocampus was then used as previously described. Inbrief, a metallic grid was placed in alignment with the mouse skull'ssutures, which were visible through the intact scalp of the mouse afterhair removal. The left hippocampus was localized by identifying thesagittal suture and then moving 2.5 mm to the left of that suture andsubsequently 3 mm in depth from the top of the skull. The grid wasremoved immediately after targeting was completed and prior to FUSapplication as to prevent interference with the sonication. Four targetsonication locations were identified relative to the sutures. The firsttarget overlapped the medial portion of the hippocampus, the lateralportion of the thalamus, and the posterior cerebral artery (PCA). Thetransducer was then moved 1 mm anterior and 1 mm lateral for the secondtarget and then 1 mm posterior for the third. The fourth and finaltarget was 1 mm medial and 1 mm anterior. In the end, four differentlocations were targeted at the corner of a 1 mm×1 mm square.

Sonication Protocol

BDNF

Definity® microbubbles (diameter: 1.1-3.3 μm, vial concentration:1.2×1010 bubbles/mL; Lantheus Medical Imaging) were composed ofoctafluoropropane gas encapsulated in a lipid shell were diluted (1:20)in phosphate-buffered saline (PBS) and then administered into the tailvein (final administered concentration: 50 μl/kg of body mass). Thisdosage is exemplary, however, it is recognized that different bubbleconcentrations can be used in accordance with the disclosed subjectmatter. For example, a clinical dose, which can be about five timeslower, can be used in accordance with the disclosed subject matter. Oneminute after injection, pulsed-wave FUS (peak-rarefactional pressure:0.46 MPa; pulse repetition frequency: 10 Hz; pulse length: 20 ms) wasapplied. Each of the four target locations was sonicated twice,resulting in a total of 8 sets of 30 s sonication with a 30 s delaybetween each set.

GDNF and NTN

The sonication parameters are provided in Table 3. All other parametersused were the same as in the case of BDNF.

Administration, Perfusion, and Sectioning

A bolus injection of BDNF compound via the femoral vein was followed 10minutes after sonication (40-90 mg/kg of mouse body mass in 0.2 ml PBS).Except for one animal that died 3 minutes after injection and wasperfused immediately, the rest of the animals were sacrificed 20-30minutes after the injection to allow for adequate circulation. Theanimals were transcardially perfused with phosphate buffered saline (4-5min.) and 4% paraformaldehyde (7-8 min) at a flow rate of 6.8 ml/min.Next, the skulls were removed and immersion-fixed for 24 hours beforeextracting the brains. Extracted brains were fixed again in 4%paraformaldehyde for 24 hours, and transferred to 10% (30 min), 20% (60min), and 30% (24 hr) sucrose solution for cryoprotection. Brain sampleswere then embedded in an Optimal Cutting Temperature (OCT) medium andwere frozen using dry ice and isopentane. Frozen blocks were sectionedhorizontally at 10-150 μm thickness for fluorescent imaging and at 5-10μm thickness for immunohistochemistry. Slices covering the entirehippocampus were selected, placed on a slide, and stored in −18° C.freezer for later analysis. In each case, the right hippocampus is notsonicated and therefore serves as the control to the left hippocampus,which is sonicated.

Immunohistochemistry

Immunohistochemistry was performed only in the case of BDNF. Fiveprimary antibodies were used: two against phosphorylated TrkB receptor(p-Y816 rabbit polyclonal [ab75173] and p-Y515, rabbit polyclonal[ab51187]) purchased from Abeam Inc. (Cambridge, Mass.), and threeagainst phosphorylated Akt (p-S473 rabbit monoclonal [#4060]),phosphorylated MAPK (p-T202/T204 rabbit monoclonal [#4370]), andphosphorylated CREB (p-S133 rabbit monoclonal [#9198]), all purchasedfrom Cell Signaling Technology (Danvers, Mass.). Slides containing thinfrozen sections (5-10 μm) were dried and placed in a citrate buffer (pH6.0) for antigen retrieval using a microwave. Slides were allowed tocool for 20 minutes prior to a PBS rinse (3×5 min) and then incubated in0.3% hydrogen peroxide in PBS (five min) to block endogenous peroxidaseactivity. Slides were washed again in PBS (3×5 min) and blocked in 10%normal goat serum with 0.1% BSA for 20 minutes. After blocking solutionwas removed, the primary antibodies were diluted in DAKO antibodydiluent solution (1:50-1:300) and incubated for 60 minutes at roomtemperature. Slides were washed in PBS for five min and incubated withbiotinylated secondary antibody (goat anti-rabbit 1:200; VectorLaboratories, Burlingame, Calif.) for 30 minutes at room temperature.Slides were washed again in PBS (3×5 min) and VECTASTAIN® ABC reagentwas added to the sections for 30 minutes (A: 1:60, B: 1:60 in PBS mixed30 min prior to use). Slides were washed in PBS (3×5 min) and peroxidasesubstrate solution DAB (DAKO, Carpinteria, Calif.) was added to sections(1 drop of DAB in 1 ml buffer). Slides were immersed in dH2O as soon ascolor developed. Sections were counterstained with hematoxylin, clearedand dehydrated with alcohols and xylene, and covered with Permount™mounting medium (Thermo Fisher Scientific, Inc Waltham, Mass., U.S.A.),and a glass coverslip.

Bright-Field and Fluorescent Microscopy

Bright-field and fluorescent images were acquired using a light andfluorescence microscope (BX61; Olympus, Melville, N.Y., USA) with afilter set at excitation and emission wavelengths of 595 nm and 615 nm,respectively.

Quantification

Bright field images were white corrected with same correction for bothright (control) and left (sonicated) images. Diaminobenzidine (DAB)stain density was then extracted from the bright field images using acolor deconvolution method and implemented in Matlab (Mathworks, Natick,Mass.) by the Open Microscopy Environment project (OME,www.openmicroscopy.org). H&E DAB built-in vectors were used for thedeconvolution step. Where DAB is uptaken, the cellular region inquestion turns brown. For each image, the mean stain intensity wascomputed using the logarithm of the stain intensity. Image artifacts(folded tissue, holes or stain droplets) were manually segmented in eachimage and removed from the analysis. For each mouse and each antibody,the percentage change (PC) of the stain intensity between the left andthe right sides was then computed as follows: PCI=100[(ILeft−IRight)/IRight]

Statistical Analysis

Statistical analysis was performed using a two-tailed Student's t-testto determine whether the BDNF concentration is significantly increasedin the sonicated (left hippocampus) region compared to the BDNFconcentration in the unsonicated (right hippocampus) region. A p<0.05was considered significant in all comparisons.

Results

FIG. 14(a) shows a fluorescent image of a 100-micron frozen brainsection from a mouse that was sacrificed 20 min after sonication. Thesonicated hippocampus (left) shows much higher fluorescent intensitythan the un-sonicated hippocampus (right), depicting blood-brain barrieropening and the extravasation of fluorescent-tagged (Alexa Fluor 594)BDNF in the sonicated region; FIG. 14(b) shows a 5-micron frozen sectionfrom the same mouse was immunohistochemically stained using a primaryantibody against phosphorylated MAPK (pMAPK). Consistent with thefluorescent image in FIG. 14(a), the intensity of DAB staining is muchgreater in the left sonicated hippocampus compared to the right control;the black box shows the enlarged area in FIG. 14(c), whereimmunoreactivity to pMAPK is shown in mossy fiber terminals (arrowhead),suprapyramidal CA3 dendrites (black star), and the axons of the Schaffercollateral system (hollow star); FIG. 14(d) shows immunohistochemicalstaining of a 5-micron frozen section from a mouse that was sacrificed 3min after sonication; the same primary antibody against pMAPK was used.No difference in DAB intensity is shown between the sonicated and thecontrol hippocampus, i.e., the 3-min case did not show significantlygreater immunoreactivity to MAPK in the sonicated region in contrast toall three 20-30 min mice that did in FIGS. 14(b)-(c); FIG. 14(e) showsnegative control performed at the same time and for the same mouse as inFIG. 14(a); no primary antibody (against pMAPK) was added to this5-micron frozen section during the staining procedure. Allmagnifications are 40× and scale bars are 500 μm except for FIG. 14(c),which is 100× and 200 μm, respectively.

FIG. 14(a) shows the diffusion of BDNF at the sonicated region in theleft hippocampus as detected by fluorescent intensity of Alexa Fluor 594(mouse sacrificed 20 min after BDNF injection). A difference can beshown in fluorescent intensities between the sonicated hippocampus(left) and the control un-sonicated hippocampus (right). Regions ofgreater intensity included parts of the thalamus, the transversehippocampal artery and its branches inside the hippocampus, the neuronsin the pyramidal (CA1-CA3) layers of the hippocampus proper, and theneurons in the hilus and granular layers of the dentate gyrus. FIGS.14(b)-(c) depict the extent of immunoreactivity to phosphorylated MAPK(activated molecule downstream of BDNF signaling; discussed below) in aDAB-stained section that was sectioned ˜300 μm dorsally from the frozensection imaged in FIG. 14(a). The DAB-stained regions closely matchedthe areas of BDNF diffusion, providing a multi-modality confirmation ofBDNF delivery across the BBB. Similar DAB intensity was observed in thecase of the mouse sacrificed 3 min after sonication (FIG. 14(d)) and thenegative control for the case in FIG. 14(a), i.e., no primary antibodyadded (FIG. 14(e)).

To demonstrate post-delivery bioactivity of the BDNF compound,immunohistochemical techniques were utilized to detect activateddownstream signaling molecules using two primary antibodies against theactivated TrkB receptor (pTrkB Y816 and pTrkB Y515), and three primaryantibodies against the phosphorylated MAPK (T202-T204), phosphorylatedCREB (S133), and phosphorylated Akt (S473). The relative activation ofthese signaling molecules in sonicated vs. non-sonicated hippocampi ofboth BDNF-administered (N=3) and control mice (N=3) were quantified bymeasuring the DAB stain intensity as described herein.

FIGS. 15(a)-(d) show immunohistochemical staining of 5-micron frozensections using a primary antibody against phosphorylated TrkB 816 (FIGS.15(a)-(c)) and a primary antibody against phosphorylated TrkB 515 (d).Mice were sacrificed 20-30 min (FIGS. 15(a)-(b), (d)) or 3 min (FIG.15(c)) after sonication. The difference in DAB intensity between thesonicated hippocampus (left column) and the contralateral controlhippocampus (right column) is detectable in all the sections (FIGS.15(a)-(d)). In FIGS. 15(a) and 15(b), the 20-30 min mice show increasedDAB intensity in the presence of TrkB 816, although, in contrast to MAPKand CREB, a lower proportion of the sections showed notable differencesbetween the left and right hippocampus. FIG. 15(b) shows a difference inthe DAB intensity in the choroid plexus of left vs. right. hippocampusgiven that the former is stained in brown (examples of DAB staining areindicated by the arrows) while the latter is only stained in blue. InFIG. 15(c), the 3-min-circulation case shows increased DAB intensity inthe presence of TrkB 816. The immunoreactivity to pTrkB is shown at theplasma membrane of neuronal cells in CA1 region (arrows in FIG. 15a ),ependymal cells of choroid plexus (arrowheads in FIG. 15b ), neuronalcells in hilus and granular layers of dentate gyrus (arrowheads in FIG.15c ), and at the plasma membrane of pyramidal neurons (arrow in FIG.15d ) and axons (stars in FIG. 15 d). Magnifications and scale bars are400× and 50 μm (FIG. 15(b)), 200× and 100 μm (FIGS. 15(a), (c)), and100× and 200 μm (FIG. 15(d)), respectively.

FIGS. 16(a)-(f) show immunohistochemical staining of 5-micron frozensections using primary antibodies against phosphorylated Akt (FIGS.16(a)-(b)) and phosphorylated CREB (FIGS. 16(c)-(e)). Mice weresacrificed 20 min (FIGS. 16(a), (c)-(d)) or 3 min (FIGS. 16(b),(e))after sonication. The box in FIG. 16(c) shows the enlarged area in FIG.16(d). Difference in DAB intensity between sonicated regions (leftimages) and the contralateral control (right images) is observable inthe 20 min samples (FIGS. 16(a), (c)-(d); black stars). The greaterintensity of the DAB stain in the sonicated region is especiallynoticeable in the thalamus in the case of pAkt (stars in FIG. 16(a)),and in the CA1 region of hippocampus (the left two stars in FIG. 16(c)),and in neuronal cells of the hilus and granular layers of dentate gyrusin the case of pCREB (arrows in FIG. 16(d), left image). Contrast thesefindings with the minimal or lack of DAB staining in neuronal cells ofthe hilus and granular layers of dentate gyrus in the controlun-sonicated hippocampus (arrows and arrowheads in FIG. 16(d), rightimage, respectively). Magnifications and scale bars are 40× and 500 μmin FIG. 16(c), 100× and 200 μm in FIGS. 16(a)-(b) and (e), and 200× and100 μm in FIG. 16(d), respectively. In FIG. 16(f), immunohistology stainintensity analysis shows percentage change between the left (FUS) andthe right (no FUS) sides of the mice brains. A significant difference(p<0.05, N=3; depicted by asterisks) was found between the BDNFadministered animal group and the control (no BDNF) animal group for theTrkB, MAPK, and CREB antibodies. Bars represent mean standard deviation.

Across all BDNF-administered mice (sacrificed 20-30 minpost-sonication), immunoreactivity to each activated signaling moleculedisplayed a distinct characteristic stain that was unique and easilyidentifiable among various brain slices. FIGS. 15(a)-(d) demonstrate thegreater presence of immunoreactivity to pTrkB Y816 (FIGS. 15(a)-(c)) andpTrkB Y515 (FIG. 15(d)) in the sonicated brain regions (left column)compared to the non-sonicated contralateral regions (right column). Onthe sonicated side, DAB staining can be seen on neuronal cell membranesin the hilus and granular layers of the dentate gyms (FIG. 15(c)), CA1(FIG. 15(a)) and CA3 (FIG. 15(d)) regions of the hippocampus, and on theependymal cell membranes of the choroid plexus in the adjacent lateralventricle (FIG. 15(b)). Such difference in DAB intensity was notapparent in all the sections, but only in some, probably due to loss ofTrkB phosphorylation state after the 20-30 min delay in sacrificing themice post-sonication and BDNF injection. On the other hand, there wasstrong immunoreactivity to pMAPK and pCREB in the sonicated lefthippocampus of BDNF-administered mice in most of the sections analyzed20-30 min post-injection. The temporal sequence of phosphorylation indownstream signaling molecules can be demonstrated when the results arecompared to those of the mouse expiring 3 min post-sonication. In those3-min cases, the difference in DAB intensity between the left sonicatedhippocampus and the control was observed in the case of the pTrkBantibody (FIG. 15(c)), but not in pMAPK (FIG. 14(d)), pAkt (FIG. 16(b)),and pCREB (FIG. 16(e)) cases, demonstrating that the BDNF-mediatedphosphorylation of the TrkB receptor is faster compared tophosphorylation of downstream molecules. As described herein,immunoreactivity to downstream molecules was more pronounced, but alsounique and distinct in the sonicated left hippocampus of >20 minsamples. Phosphorylated MAPK was detected in axons and dendrites of thepyramidal and granular neurons, but not in the neuronal cell bodies.pMAPK immunoreactivity was present in the mossy fibers of the CA3hippocampal neurons (FIGS. 14(b)-(c)). The phosphorylated CREBimmunoreactivity was observed in the nuclei and cytoplasm of theneuronal soma in all CA regions and layers of the sonicated hippocampus.With respect to the right (unsonicated) hippocampus (in both the controland BDNF-administered cases), lower levels of pMAPK and pCREBimmunoreactivity were detectable (FIGS. 14(b)-(c) and FIGS. 16(c)-(d),right columns). In the case of pCREB, however, the CA1 region and partsof the granular layers of the dentate gyms in the right unsonicatedhippocampus showed absence of immunoreactivity (FIGS. 16(c)-(d), rightcolumns).

FIGS. 17(i)(a) and (ii)(a) show T1-weighted MR image of the entire mousehead verifying BBB opening using gadolinium enhancement and FIGS.17(i)(b) and (ii)(b) show fluorescence image magnified in the region ofinterest where the highest gadolinium enhancement was detected (whiterectangle) in two separate murine brains, one with fluorescently tagged(Alexa Fluor) GDNF (Invitrogen, Inc.) in FIGS. 17(i)(a)-(b) and theother with Neurturin (courtesy of Judith P. Golden, Ph.D., WashingtonUniversity Medical Center and Invitrogen, Inc.) in FIGS. 17(ii)(a)-(b),were systemically administered with the target being the caudate putameninstead of the hippocampus.

According to the statistical results, more significant cascade effectswere shown to be triggered in the sonicated regions compared to thecontralateral (control) side in all cases, indicative of the fact that acritical BDNF concentration was reached sufficient of triggering theseeffects. Also, two additional neurotrophic factors that have shownpromise in treating Parkinson's disease (both involved in clinicaltrials), were also tested, namely the glia-derived neurotrophic factor(GDNF) (FIGS. 17(i)(a)-(b)) and neurturin (NTN) (FIGS. 17(ii)(a)-(b))using the same sonication parameters as in the BDNF study but targetingthe caudate putamen instead of the hippocampus in two mice as thatregion is more relevant to Parkinson's. It can be shown that, althoughall these three proteins can be of similar molecular weight and overallconsistency, not all neutrotrophic factors cross the blood-brain barrierafter opening with FUS. Neurturin may behave similarly to BDNFpermeating through the opened barrier and into the parenchyma (FIGS.17(ii)(a)-(b)); however, GDNF does not (FIGS. 17(i)(a)-(b)). The latterfinding was shown in ten mice while the former in two. There may befewer receptors in the brain for GDNF than for BDNF, i.e., the twoproteins on two different transport mechanisms. Also, the GDNF may bebroken down in circulation within the first 45 s and upon imagingpost-mortem no fluorescence was found in the brain (FIG. 17(ii)(b)),kidneys, liver, bladder or testes. Therefore, GDNF may be undetectablein the brain parenchyma but may also be absent from other organs,indicating potential differences in the GDNF systemic administrationcompared to those of BDNF and NTN.

In the case of antibodies against pTrkB (Y515 and Y816 resultscombined), pMAPK, and pCREB, mean percent changes in DAB stain intensitybetween the left and right hippocampal regions were greater in theBDNF-administered mice (25.22, 60.58, and 56.91%) compared to those inthe control mice (−1.36, −9.20, and −8.67%), respectively (p<0.05; FIG.16(f)). In the case of the pAkt antibody, the mean percent changes inthe DAB stain intensity of the BDNF-administered mice and the controlmice were similar, although parts of the sonicated thalamus in theBDNF-administered mice showed greater DAB stain intensity than theun-sonicated side in a few sections (FIG. 16(a)).

Thus, the brain-derived neurotrophic factor (BDNF) can cross theultrasound-induced blood-brain barrier opening, and can also triggersignaling pathways in the pyramidal neurons of mice in vivo from themembrane to the nucleus. As shown with two additional neurotrophicfactors, namely GDNF and NTN, these findings can depend on thepharmacokinetics and other properties of the molecular uptake of themolecule in the brain. However, as the molecule after permeation throughthe opened BBB triggered a molecular cascade and entered the neuronalnucleus, focused ultrasound in conjunction with microbubbles cangenerate downstream effects at the cellular and molecular level and thusincrease the drug's efficacy and potency in controlling or reversingdisease.

Example 3

In another exemplary experiment, the dependence of both the spatialextent and the duration of FUS-induced BBB opening in vivo withdifferent microbubble sizes and peak-rarefactional pressures (PRPs) isshown. Gd-DTPA-BMA retention in the brain parenchyma can be used as asignature of the area of BBB opening. Volumetric quantification of theregion of BBB opening can be assessed by measuring the diffusion volumeof Gd-DTPA-BMA in the sonicated region of the brain, for example theright hippocampus, detected by the longitudinal signal enhancement. Inthe experiment, starting with the day of sonication and continuing up to5 days following sonication, pre and postcontrast enhancementT1-weighted high resolution MR images were consecutively acquired ateach timepoint.

Materials and Setup

Ultrasound Setup

Acoustic waves used were generated by a single-element,spherical-segment FUS transducer (center frequency: 1.5 MHz, focaldepth: 60 mm, radius: 30 mm; Imasonic, France), which was driven by afunction generator (Agilent, Palo Alto, Calif.) through a 50-dB poweramplifier (E&I, Rochester, N.Y.) (for example as shown in FIG. 5). Acentral-void (radius: 11.2 mm) of the therapeutic transducer held apulse-echo ultrasound transducer (center frequency: 10 MHz, focallength: 60 mm), which was used for imaging, with their two foci aligned.The imaging transducer was driven by a pulser-receiver (Olympus,Waltham, Mass.) connected to a digitizer (Gage Applied Technologies,Lachine, QC, Canada). A cone filled with degassed and distilled waterwas mounted onto the transducer system and was fitted with apolyurethane membrane (Trojan; Church & Dwight Co., Princeton, N.J.).The transducers were attached to a computer-controlled 3D positioningsystem (Velmex, Lachine, QC, Canada). The targeting procedure has beendescribed herein. For example, the FUS transducer was moved 3 mmlaterally of the sagittal suture and 2 mm anterior of the lambdoidsuture. A needle hydrophone (Precision Acoustics, Dorchester, Dorset,UK, needle diameter: 0.2 mm) was used to measure the three-dimensionalpressure field in a degassed water-tank prior to the in vivoapplication. The FUS focal spot overlapped with the right hippocampusand the latter portion of the thalamus, since the axial and lateralfull-widths at half-maximum intensities of the beam were 7.5 mm and 1mm, respectively. The left hippocampus fissure was used as a control,and was not sonicated. Pulsed FUS was emitted for 60 s, with a burstrate of 10 Hz, 100 burst cycles, at acoustic pressures adjusted tocorrespond to 0.30, 0.45, and 0.60 MPa (peak-rarefractional), afteraccounting for 18% murine skull attenuation. These pressures wereobtained experimentally in degassed water. The mice were anesthetizedusing 1.25-2.50% isoflurane (SurgiVet, Smiths Medical PM, Wisconsin)mixed with oxygen during FUS.

Size Isolated Microbubbles

The microbubbles used were size-isolated from a poly-dispersedmicrobubble distribution using differential centrifugation. The bubbleshad a 1,2-disearoyl-sn-glycero-3-phosphocholine (DSPC) andpolyoxyethylene-40 stearate (PEG40S) lipid shell and perfluorobutane(PFB) core. After the centrifugation and resuspension processes wererepeated several times, three desired ranges of 1-2, 4-5, and 6-8 mm indiameter were isolated. A bolus of 1 mL/g at a concentration of 8×108/mLwas injected intravenously through the tail vein prior to sonication.

Magnetic Resonance Imaging

BBB opening in the murine hippocampus was confirmed using a 9.4 T system(Broker Medical; Boston, Mass.). All mice were anesthetized orally using1-2% of isoflurane mixed with oxygen and were placed inside thevertical-bore, having a fixed position in a plastic tube with a 3.0 cmdiameter birdcage coil. Vital signals were monitored and respirationrate was approximately 55 breaths/min. Each MRI session included a preand a postcontrast enhancement, T1-weighted 2D FLASH acquisition (TR/TE:230/3.3 ms, flip angle: 70°, NEX: 18, resolution 86 mm×86 mm, slicethickness: 500 mm, 23 slices, F.O.V.: 22 mm×16.5 mm, matrix size256×192, receiver bandwidth: 50 kHz), obtained respectively 15 min aftersonication and 55 min after injection of the MRI contrast agent(Gd-DTPA-BMA). Signal enhancement in the area of the sonicatedhippocampus reached a peak approximately 1 h after IP injection.Therefore, the CE-T1 images were acquired 55 min following theinjection. Omniscan™ (Gd-DTPA-BMA) was used to enhance the MR contrastin the murine brain, as a tracer for the BBB opening since it candiffuse into the brain parenchyma following BBB's altered permeabilityin the targeted area. Gd-DTPA-BMA was administered intraperitoneally(IP) at a dose of 6 mmol/kg. An IP bolus can provide more temporallyconsistent and sustained MR enhancement than an IV bolus and can belogistically simpler. Preliminary work on the IP bolus dosage indicatedthat the 6 mmol/kg can avoid the signal decrease which can be observedwhen, due to excessive Gd-DTPA-BMA, the T2 relaxivity dominates the T1relaxivity. The same MRI session was repeated daily starting from theday of the BBB opening (day 0) and lasting up to 5 days. In the fivecases where signal enhancement was detected on day 5, MRI sessions wererepeated on day 7.

Animal Preparation

All procedures used in this study involving animals were approved by theColumbia University Institutional Animal Care and Use Committee. A totalof forty-two (n=42) wild-type mice, (strain C57BL/6, mass: 20-25 g, sex:male, Harlan, Indianapolis, Ind.) was used for this experiment,separated into ten groups. Each mouse in the first nine groups wassonicated with a different combination of PRP, i.e., 0.30, 0.45, or 0.60MPa, and a microbubble diameter, i.e., 1-2, 4-5, or 6-8 mm. These miceunderwent MRI for several days after FUS. A sham group had three (n=3)mice, for which the whole procedure was repeated without FUS, i.e.,anesthesia, injection of microbubbles, and MRI sessions for up to 5days.

Histology and Imaging

On day 7, all mice were euthanized and transcardially perfused with 30mL PBS and 60 mL 4% paraformaldehyde. Brains were soaked inparaformaldehyde for 24 h. Skulls were removed, and the brains werefixed again in 4% paraformaldehyde for 6 days, followed by conventionalpostfixation procedures. The paraffin-embedded specimens were sectionedhorizontally at a thickness of 6 mm. 24 sections were stained andexamined for each brain. Sections were stained with hematoxylin andeosin and then examined for red blood cell extravasations into the brainparenchyma as well as cell loss.

Image Processing and Volumetric Measurements

The enhancement in CE-T1 MR images was quantified in ellipticcylindrical VOIs encompassing the hippocampal formation, at twocontralateral regions, in the right (sonicated) and the left (control)hemisphere, on each brain for each day. The major diameter of theelliptic cylinders was 4.3 mm, the minor diameter 3.4 mm, and the height4.5 mm, covering an area of approximately 14,000 voxels from a total ofnine consecutive horizontal slices, on each side. Therefore, the totalvolume on each cylinder was 52 mm³. In order to quantify the BBB openingvolume at the sonicated side, an intensity threshold was determined andthe contrast-enhanced pixels in the vessels and ventricles wereexcluded. To address these issues, the signal intensity was averagedover a small circular region of 1 mm in diameter, centered around thenonsonicated contralateral side close to the hippocampus and used as areference. The number of voxels in the right (sonicated) and left(control) VOI at an intensity of 2.5 standard deviations (S.D.) or abovethe reference were counted. The total number of these voxels in the leftVOI was then subtracted from the respective total number of voxels inthe right VOI, to exclude volume contrast-enhancement in the vessels andventricles. Precontrast images were used for the detection of anyhyperintense areas before Gd-DTPA-BMA injection, and were notcoregistered with the postcontrast.

The measurements in the sham group were used as the baseline, denotingthat the integrity of the BBB was fully restored. Following thecalculation of the mean and standard deviation (S.D.) of the volumetricmeasurements for each group, i.e., mice sonicated at the same PRP andmicrobubble size, a two-tailed Student's t-test was performed, and if nostatistically significant difference compared to the control baselinewas observed (P>0.05), then the BBB was considered to have closed. Inother words, the closing criterion was checked on each day for eachgroup and not for each measurement individually. All groups met theclosing criterion by day 5. For the five individual cases where signalenhancement was shown on day 5, MRI was repeated on day 7. Not all ofthese cases however belonged to the same group, and the groups theybelonged to could still meet the closing criterion based on thestatistical analysis.

In order to show the differences in the timeline for closing betweenmicrobubble sizes and pressures, a statistical analysis was performed onthe volume of diffusion of Gd-DTPA-BMA on day 0. To evaluate the effectof the microbubble size, a two-tailed Student's t-test was performed foreach PRP, i.e., between 1-2 mm and 4-5 mm, between 1-2 mm and 6-8 mm, aswell as between 4-5 mm and 6-8 mm. Differences between the differentgroups regarding the volume of BBB opening and the time required for theopened BBB to be reinstated were examined.

Results

FIG. 18 shows horizontal consecutive CE-T1 images (500 mm thickness,dorsal on top, ventral on bottom) from day 0 to 3 for a 6-8 mm/0.30 MPacase. The BBB opening reduced radially towards the center of the FUSbeam over time, until closing was detected in day 3, when no Gd-DTPA-BMAdiffused from the vasculature to the brain parenchyma.

An example of the volume quantification can be shown in FIG. 18. TheVOIs were manually traced to overlap with the right and left hippocampi.On both sides, voxels with an intensity 2.5 S.D. or above the referenceintensity are overlaid in red. As shown in these images, when there isno diffusion of Gd-DPTA-BMA from the vasculature to the brainparenchyma, e.g., on the control side, the vessels and the ventriclescan have CE-T1 intensity above the threshold. However, when thepermeability of the BBB is altered on the right hippocampus as a resultof the FUS, Gd-DTPA-BMA can diffuse in that region and the area ofopening can be overlaid in red. The example in FIG. 18 shows the samebrain in multiple 2D slices where 6-8-mm bubbles at a PRP of 0.30 MPawere used. The volume of opening was reduced radially towards the focalregion over several subsequent days, until no trans-BBB diffusion wasdetected on day 3, signifying that the BBB was successfully reinstated.

FIG. 19 shows coronal reconstructions from horizontal CE-T1 images withone example provided in each case of PRP and microbubble size. FIGS.20(a)-(c) show volume of diffusion of Gd-DTPA-BMA area, depicting BBBopening, for PRP of 0.30, 0.45, and 0.60 MPa and the sham group withmicrobubbles of 1-2 mm in FIG. 20(a), 4-5 mm in FIG. 20(b), and 6-8 mmin FIG. 20(c) in diameter. Error bars correspond to standard deviationand (*) denotes closing. FIG. 21 shows volume of BBB opening on day 0versus time to closing, showing that the duration of BBB openingincreased monotonically with the volume of opening on day 0.

For all cases of microbubble sizes and pressures studied, the BBB wasfound to be reinstated by day 5, and the duration of opening depended onthe microbubble diameter and PRP used. In FIG. 19, reconstructions ofthe horizontal planes, sliced coronally at the level of the hippocampalformation, at all pressures and microbubble sizes are shown. In FIG. 19,it can also be shown that the area of Gd-DTPA-BMA diffusion in the brainand its spatial characteristics can depend on the microbubble size andpressure used. The BBB permeability to Gd-DTPA-BMA can occur dorsally inthe brain in the cases when larger microbubbles were used, i.e., not inthe 1-2 mm case, where diffusion of the contrast agent Gd-DTPA-BMA wasobserved mainly ventrally in the brain near the vasculature. This effectcan be shown further at higher PRPs, i.e., 0.60 MPa. Volumetricmeasurements are shown in FIGS. 20(a)-(c) for the 1-2, 4-5, and 6-8 mmmicrobubble cases. At 0.30 MPa with the 1-2 mm microbubbles no BBBopening was detected. Depending on the microbubble and pressure used,the BBB closing occurred within 24 h and 5 days after sonication. Morespecifically, with the 1-2 mm microbubbles (FIG. 20(a)), closing wasfound to be closed on day 1 and 2 at 0.45 and 0.60 MPa, respectively.With the 4-5 mm and 6-8 mm microbubbles, the BBB was found to be closedon day 2 at 0.30 MPa, day 3 at 0.45 MPa, and day 5 at 0.60 MPa. Aproportional relationship between the volume and the duration of the BBBopening regardless of the PPR and microbubble size used to induce theopening can thus be shown (for example in FIG. 21). Linear regressioncan show a correlation of R2=0.72. Excluding day 0 a logarithmic fitindicated a correlation of R2=0.78.

The results of the statistical analysis on day 0 are shown in Table 4.At 0.30 MPa, opening was induced only in the 4-5 and 6-8 mm cases,without statistically significant difference (P>0.05) between these twodiameter ranges (Table 4), and the BBB was restored by day 2 in bothcases. At 0.45 MPa, the volume of BBB opening induced with 1-2 mmmicrobubbles, was statistically different (P<0.01) than that with largermicrobubbles (Table 4), and occurred within 24 h, compared to 2-3 daysneeded in the cases of the larger microbubbles. Finally, in Table 4, itcan be shown that at 0.60 MPa, there was a statistically significantdifference between the 1-2-mm and 6-8-mm bubbles (P<0.05), and at least2 days were required for the BBB to be reinstated in the 1-2 mm case, asopposed to 4-5 days required for the larger microbubbles.

FIGS. 22(a)-(d) show examples of one out of the five cases where damagewas detected. FIGS. 22(a)-(b) are horizontal precontrast T1-weightedimages, acquired on day 0 and 7, respectively. Hyperintensity can beshown (white arrows) on the sonicated side, more enhanced on day 7.FIGS. 22(c)-(d) are the H&E stained slices, magnified (10) at the left(control) hippocampus and at the right (sonicated) hippocampus,respectively. In FIG. 22(c) cell loss can be detected at the dentategyrus and CA1 area (white arrows).

Correlation can also be found between all histological damage cases andhyperintensity in the precontrast T1-w images. An example is shown inFIG. 22, where hyperintensity was detected at the sonicated region inthe precontrast image, and cell loss was detected on the H&E stainedslices at the level of the hippocampus. Damage was observed uponhistological examination only in five animals, approximately 13% of thetotal number of animals used, all of which were at higher PRPs.

Discussion

In this Example, the reversibility of BBB opening was investigated usingGd-DTPA-BMA that cannot cross the BBB when the BBB is reinstated orclosed. Spatial (i.e., volume) and temporal (i.e., duration)characteristics of the intact or BBB's altered function were analyzed,while opening was induced using three different microbubble sizes andthree different PRPs. BBB opening induced by FUS can be shown to betransient, but the duration can depend on the acoustic parameters andbubbles used. Also, the BBB opening can be dependent on the acousticpressure used as well as the microbubble size. The features of the BBBself-repairing characteristic were analyzed under a combination ofdifferent acoustic parameters, and a range of mono-dispersedmicrobubbles. A proportional relationship between the BBB opening volumeand the time required for closing can be shown. Therefore, both the BBBopening volume and duration was shown to be dependent on the acousticpressure and the microbubble size used.

The spatial characteristics of the BBB opening and its reversibility wasshown as follows. Firstly, as seen in the examples of FIGS. 18 and 19,the BBB function can be reinstated in a reverse direction to that of thediffusion after opening, i.e., closing starts from the outer openedregions and ends at the focal region while also being dependent on thehippocampal vasculature. It was also shown that the permeability valuestowards the center of the focal region were higher. The peak of theacoustic pressure distribution can lie at the center of the focal spotwhich can result in larger BBB openings, hence taking longer to close.Additionally or alternatively, where the vasculature is denser, therecan be a higher number of opening sites, and thus longer timelines forclosing to be completed.

Even though the volume of opening induced by the 6-8-mm bubbles can begreater than the volume induced by the 4-5 mm microbubbles, these twomicro-bubble sizes did not differ significantly in this experiment (asshown in Table 4) and the days required for closing were also similar(FIGS. 20(b),(c)). The differences can be relatively small because both4-5-mm and 6-8-mm bubbles can be within the diameter size range of thecapillary, i.e., 4-8 mm, and therefore they can both be in contact withthe capillary wall, exerting forces on it while being acousticallydriven. However, the mechanical stress on the capillary walls due to theacoustically driven microbubbles for a specific pressure when induced bythe 6-8-mm can be larger than when induced by the 4-5-mm bubbles,because higher PRP can reach a similar size expansion as the 6-8-mm andhave a similar effect, hence the BBB opening volume can be increased.

TABLE 4 Statistical Significance of the Volume of Diffusion of Gd-DTPA 

 BMA on Day 0, Comparing Different Cases of Microbubble Diameters perPRP Day 0 1-2 (μm) 4-5 (μm) 0.30 MPa 6-8 μm — P > 0.05 0.45 MPa 6-8 μm P< 0.01 P > 0.05 4-5 μm P < 0.01 — 0.60 MPa 6-8 μm P < 0.05 P > 0.05 4-5μm P > 0.05 —

The BBB opening volume using 1-2-mm bubbles can be lower than for thelarger microbubbles, (Table 4) and the BBB can close faster. Therefore,for improved BBB recovery at high pressures, smaller microbubbles can bepreferable. The relative expansion of a microbubble can be inverselyproportional to its resting diameter. 1-2-mm bubbles can induce noopening at 0.30 MPa, where only stable cavitation is expected. Thus,stable oscillations of smaller microbubbles can be insufficient toinduce the repeated stress against the capillary wall at 0.30 MPa, butcan be adequate at 0.45 MPa, and even more so at 0.60 MPa. It can alsobe shown that fragmentation occurs more frequently for microbubbles witha relatively small rather than a large resting diameter at a thresholdsize of 2.5 mm. Below that level, fragmentation may occur and bubblessmaller than 2 mm can be fragmented prior to reaching the endotheliumand not interact with the vessel wall. Therefore, it was shown that thesmaller microbubbles can be incapable of inducing opening in sites awayfrom the bigger vessels, because they can undergo fragmentation at thebeginning of the FUS pulse before perfusing the microvasculature. Andthus, the therapeutic efficacy of 1-2 mm bubbles in the capillaries maybe decreased.

The timeline for closing can be longer than 3-24 h. This can be due tomultiple factors. First, the microbubble formulation used in thisExample could be different than the commercially available contrastagents, in terms of the combination of shell properties, gas core anddiameter size; therefore, this microbubble formulation used here can bea parameter to induce variations. However, the size range of thecommercially available microbubbles can be closer to the 1-2-mm bubblesused in this experiment, which were shown to induce BBB opening that canclose within 24 h.

At lower PRPs, the BBB can close relatively faster, and the PRPs usedcan be below 0.50 MPa. Moreover, the FUS frequency (1.5 MHz) can affectclosing time. The resonance frequency can decrease with the microbubbleradius, and can also decrease with decreasing microvessel radius.Therefore, using a lower frequency for example, which can be closer tothe resonance frequency of the micro-bubbles within the microvessles andcapillaries, the aforementioned effects can be further enhanced.Finally, the agents used to cross the BBB for the detection of openingcan have larger MWs (Magnevist®: 938Da, HRP: 40 kDa, Evans Blue: 961 Da)while it was shown that the BBB becomes less permeable at higher MWs.Since the administered Gd-DTPA-BMA can be a relatively smaller (574 Da)agent and above the size threshold to cross the BBB (400 Da), thedisclosed subject matter can have an increased sensitivity regarding thedetection of BBB opening, which can contribute to longer closing times.

In the five cases where cell loss was detected in the H&E stained brainsections on the sonicated region, hyperintensity was also detected onthe precontrast MRI images at the corresponding regions. The signalenhancement detected in these areas in the precontrast T1-w images canbe due to permanent damage, blood present in the brain or arrestedGd-DTPA-BMA in damaged vasculature, and thus BBB can not be completelyrestored.

The BBB remained opened over several days in some cases. It wasthereafter restored and 87% of the cases showed no detectable damage,while the remaining 13% showed minimal cell loss. Thus, first, atshorter burst lengths opening can be induced with less damage, andsecond, the self-repairing mechanism of the BBB can restore certaintypes of injury induced to the brain after FUS.

BBB can be disrupted longer than the MRI system used can detect and theresolution of the images can acquire. Additionally, the firstacquisition or postcontrast T1-w images can be 1 h after FUS, and withinthis time some cases with relatively small BBB opening can beundetected. Finally, the circulation times and persistence of all thedifferent sizes of microbubbles can be similar for the 10-20 s intervalbetween injection and FUS, however, differences can affect the results.

In this Example, the volume of BBB opening and the time required for theBBB to be reinstated depends on the microbubble size and the acousticpressure. In addition, the time for closing is proportional to thevolume of opening induced by FUS, and BBB can recover its functionalitybetween 24 h and 5 days after. The BBB opening volume can decreaseradially towards the center of the focal spot over time. At loweracoustic pressures, relatively smaller microbubble diameters can inducea lower volume of BBB opening, and the closing timeline can be differentthan that using larger microbubbles. As the PRP increases, thedifferences in BBB opening and closing between the different microbubblesizes can be reduced. The BBB can close relatively faster when smallmicrobubbles are used, while for the 4-5 and 6-8 mm the same durationfor closing can be shown. Finally, hyperintensity in the area of BBBopening can be detected in the precontrast MR images in the cases wheredamage was concluded in histology. The FUS-opened BBB self-repairingcharacteristics can be shown, spatially and temporally, and the systemsand methods described herein can be adjusted according to thepharmacokinetic needs of administered CNS drugs.

It will be understood that the foregoing is only illustrative of theprinciples described herein, and that various modifications can be madeby those skilled in the art without departing from the scope and spiritof the disclosed subject matter. For example, the system and methodsdescribed herein are used for opening the blood-brain barrier of asubject. It is understood that that techniques described herein areuseful for opening of other tissues. Further, the techniques describedhave been performed on mice but it is understood the techniques areapplicable to other subject, such as humans. Moreover, features ofembodiments described herein can be combined and/or rearranged to createnew embodiments.

We claim:
 1. A method for opening a tissue to a target value usingmicrobubbles, comprising; targeting a region of said tissue to open;determining at least one acoustic parameter corresponding to said targetvalue, wherein said at least one acoustic parameter is selected tocontrol a magnitude and a type of one or more acoustic cavitation eventsto open said tissue to said target value, and wherein said target valuecorresponds to a treatment and/or imaging value to allow passage ofcertain molecules and/or agents; and applying an ultrasound beam at saidat least one acoustic parameter to said targeted region such that saidtissue is opened with said microbubbles to said target value.
 2. Themethod of claim 1, further comprising positioning said microbubbles inproximity to said targeted region, wherein positioning said microbubblescomprises performing at least one injection of said microbubbles suchthat said microbubbles are positioned proximate to said targeted region.3. The method of claim 2, further comprising determining at least one ofa number of injections of said microbubbles corresponding to said targetvalue and a duration of injection of said microbubbles corresponding tosaid target value.
 4. The method of claim 2, wherein said at least oneinjection comprises at least one of a systemic injection, a bolusinjection and a slow diffusion injection.
 5. The method of claim 1,wherein controlling said one or more acoustic cavitation eventscomprises controlling a number of said one or more acoustic cavitationevents.
 6. The method of claim 1, wherein said acoustic parameter isselected from at least one of a pulse length, a pulse repetitionfrequency, a burst length, and a burst repetition frequency.
 7. Themethod of claim 1, wherein determining at least one acoustic parameterfurther comprises determining at least one of a frequency, a durationand a pressure range corresponding to said target value.
 8. The methodof claim 7, wherein the at least one acoustic parameter comprises saidpressure range, and wherein said pressure range corresponds to aresonance frequency of said microbubbles proximate to said targetedregion.
 9. The method of claim 1, further comprising determining aconcentration range of microbubbles corresponding to said target valueprior to said positioning of said microbubbles.
 10. The method of claim1, wherein said tissue comprises at least one of a vessel, a cell and ablood-brain barrier.
 11. The method of claim 1, further comprisingapplying an ultrasound beam to move said microbubbles into vessels ofsaid tissue.
 12. The method of claim 1, wherein said microbubblescomprise at least one of acoustically activated microbubbles, moleculecarrying microbubbles and microbubbles having a size range between 1 and10 microns.
 13. The method of claim 2, wherein said microbubblescomprises said molecule-carrying microbubbles, and wherein said moleculecomprises at least one of a medicinal molecule, a contrast agent, abiomarker and a liposome.
 14. The method of claim 1, further comprisingpositioning at least one of a medicinal molecule and a contrast agent inproximity to said targeted region.
 15. The method of claim 1, furthercomprising imaging said targeted region to form an image of said openedtissue, wherein imaging said targeted region comprises applying anultrasound beam to said targeted region, utilizing a magnetic resonanceimaging device to image said targeted region, or utilizing afluorescence imaging device to image said targeted region.
 16. Themethod of claim 1, wherein at least a portion of said opened tissuecloses, and the method further comprises imaging said targeted region toform an image of said closed tissue.
 17. The method of claim 16, furthercomprising reopening at least a portion of said closed tissue.
 18. Themethod of claim 1, wherein the target value comprises a measure ofincreased size of vessels in the tissue.
 19. The method of claim 1,wherein the target value comprises a target size of an opening of thetissue.
 20. The method of claim 1, wherein the target value comprises atarget rate at which molecules pass through the tissue.
 21. The methodof claim 1, wherein the at least one acoustic parameter is selected toopen the tissue to the target value using the controlled type of the oneor more acoustic cavitation events.
 22. The method of claim 21, whereinthe controlled type of the one or more acoustic cavitation eventscomprises at least one of stable cavitation and inertial cavitation. 23.A system for opening a tissue to a target value using a solution ofmicrobubbles having size range corresponding to said target value,comprising: a targeting assembly for targeting a region of said tissue;an introducer for delivering said solution to a location proximate tosaid targeted region; and a transducer, coupled to said targetingassembly, for applying an ultrasound beam to said targeted region atleast one acoustic parameter corresponding to said target value therebyopening said tissue with said microbubbles to said target value, whereinsaid at least one acoustic parameter is selected to control a magnitudeand a type of one or more acoustic cavitation events to open said tissueto said target value, and wherein said target value corresponds to atreatment and/or imaging value to allow passage of certain moleculesand/or agents.
 24. The system of claim 23, wherein said targetingassembly comprises at least one of an ultrasound transducer and one ormore members for placement on an anatomical landmark of said tissue, andwherein said system further comprises an imaging device for capturingimage data of said opened tissue of said targeted region, and aprocessor, operatively coupled to said imaging device, for processingsaid image data to form an image therefrom.
 25. The system of claim 23,wherein said solution of microbubbles further comprises a microbubblesconcentration range corresponding to said target value.
 26. A method fordelivering a drug across a tissue, comprising opening a tissue to atarget value using microbubbles, comprising: targeting a region of saidtissue to open; determining at least one acoustic parametercorresponding to said target value, wherein said at least one acousticparameter is selected to control a magnitude and a type of one or moreacoustic cavitation events to open said tissue to said target value, andwherein said target value corresponds to a treatment and/or imagingvalue to allow passage of certain molecules and/or agents; applying anultrasound beam at said at least one acoustic parameter to said targetedregion such that said tissue is opened with said microbubbles to saidtarget value; and injecting said drug into the tissue in proximity tosaid opening.
 27. The method of claim 26, wherein said tissue comprisesa blood-brain barrier.
 28. A method for opening a tissue for a targetduration of time using microbubbles, comprising: targeting a region ofsaid tissue to open; selecting microbubbles having at least onemicrobubble parameter corresponding to said target duration of time,wherein said at least one microbubble parameter is selected to control amagnitude and a type of one or more acoustic cavitation events to opensaid tissue for said target duration of time, and wherein said targetduration of time corresponds to a treatment and/or imaging value toallow passage of certain molecules and/or agents; and applying anultrasound beam to said targeted region such that said tissue is openedwith said microbubbles for said target duration of time.
 29. The methodof claim 28, wherein the at least one microbubble parameter comprises amicrobubble size.
 30. The method of claim 29, further comprisingselecting at least one acoustic parameter and applying the ultrasoundbeam at said at least one acoustic parameter, wherein said at least oneacoustic parameter is selected to further control the magnitude and thetype of the one or more acoustic cavitation events to open said tissuefor said target duration of time.